Nucleic acids encoding antibodies specifically binding to masp-3

ABSTRACT

The present invention relates to MASP-3 inhibitory antibodies and compositions comprising such antibodies for use in inhibiting the adverse effects of MASP-3 dependent complement activation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of prior application Ser. No.15/665,030, filed Jul. 31, 2017, which claims the benefit of U.S.Provisional Application No. 62/369,674, filed Aug. 1, 2016, and claimsthe benefit of U.S. Provisional Application No. 62/419,420, filed Nov.8, 2016, and claims the benefit of U.S. Provisional Application No.62/478,336, filed Mar. 29, 2017, all three of which are herebyincorporated by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is MP_1_0254_US2_Sequence_Listing_20200401_ST25;the file is 191 KB; was created on Apr. 1, 2020 and is being submittedvia EFS-Web with the filing of the specification.

BACKGROUND

The complement system provides an early acting mechanism to initiate,amplify and orchestrate the immune response to microbial infection andother acute insults (M. K. Liszewski and J. P. Atkinson, 1993, inFundamental Immunology, Third Edition, edited by W. E. Paul, RavenPress, Ltd., New York), in humans and other vertebrates. Whilecomplement activation provides a valuable first-line defense againstpotential pathogens, the activities of complement that promote aprotective immune response can also represent a potential threat to thehost (K. R. Kalli, et al., Springer Semin. Immunopathol. 15:417-431,1994; B. P. Morgan, Eur. J. Clinical Investig. 24:219-228, 1994). Forexample, C3 and C5 proteolytic products recruit and activateneutrophils. While indispensable for host defense, activated neutrophilsare indiscriminate in their release of destructive enzymes and may causeorgan damage. In addition, complement activation may cause thedeposition of lytic complement components on nearby host cells as wellas on microbial targets, resulting in host cell lysis.

The complement system has also been implicated in the pathogenesis ofnumerous acute and chronic disease states, including: myocardialinfarction, stroke, ARDS, reperfusion injury, septic shock, capillaryleakage following thermal burns, postcardiopulmonary bypassinflammation, transplant rejection, rheumatoid arthritis, multiplesclerosis, myasthenia gravis, and Alzheimer's disease. In almost all ofthese conditions, complement is not the cause but is one of severalfactors involved in pathogenesis. Nevertheless, complement activationmay be a major pathological mechanism and represents an effective pointfor clinical control in many of these disease states. The growingrecognition of the importance of complement-mediated tissue injury in avariety of disease states underscores the need for effective complementinhibitory drugs. To date, Eculizumab (Solaris®), an antibody againstC5, is the only complement-targeting drug that has been approved forhuman use. Yet, C5 is one of several effector molecules located“downstream” in the complement system, and blockade of C5 does notinhibit activation of the complement system. Therefore, an inhibitor ofthe initiation steps of complement activation would have significantadvantages over a “downstream” complement inhibitor.

Currently, it is widely accepted that the complement system can beactivated through three distinct pathways: the classical pathway, thelectin pathway, and the alternative pathway. The classical pathway isusually triggered by a complex composed of host antibodies bound to aforeign particle (i.e., an antigen) and thus requires prior exposure toan antigen for the generation of a specific antibody response. Sinceactivation of the classical pathway depends on a prior adaptive immuneresponse by the host, the classical pathway is part of the acquiredimmune system. In contrast, both the lectin and alternative pathways areindependent of adaptive immunity and are part of the innate immunesystem.

The activation of the complement system results in the sequentialactivation of serine protease zymogens. The first step in activation ofthe classical pathway is the binding of a specific recognition molecule,C1q, to antigen-bound IgG and IgM molecules. C1q is associated with theC1r and C1s serine protease proenzymes as a complex called C1. Uponbinding of C1q to an immune complex, autoproteolytic cleavage of theArg-Ile site of C1r is followed by C1r-mediated cleavage and activationof C1s, which thereby acquires the ability to cleave C4 and C2. C4 iscleaved into two fragments, designated C4a and C4b, and, similarly, C2is cleaved into C2a and C2b. C4b fragments are able to form covalentbonds with adjacent hydroxyl or amino groups and generate the C3convertase (C4b2a) through noncovalent interaction with the C2a fragmentof activated C2. C3 convertase (C4b2a) activates C3 by proteolyticcleavage into C3a and C3b subcomponents leading to generation of the C5convertase (C4b2a3b), which, by cleaving C5 leads to the formation ofthe membrane attack complex (C5b combined with C6, C7, C8 and C-9, alsoreferred to as “MAC”) that can disrupt cellular membranes resulting incell lysis. The activated forms of C3 and C4 (C3b and C4b) arecovalently deposited on the foreign target surfaces, which arerecognized by complement receptors on multiple phagocytes.

Independently, the first step in activation of the complement systemthrough the lectin pathway is also the binding of specific recognitionmolecules, which is followed by the activation of associated serineprotease proenzymes. However, rather than the binding of immunecomplexes by C1q, the recognition molecules in the lectin pathwaycomprise a group of carbohydrate-binding proteins (mannan-binding lectin(MBL), H-ficolin, M-ficolin, L-ficolin and C-type lectin CL-11),collectively referred to as lectins. See J. Lu et al., Biochim. Biophys.Acta 1572:387-400, (2002); Holmskov et al., Annu. Rev. Immunol.21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000)). See alsoJ. Luet et al., Biochim Biophys Acta 1572:387-400 (2002); Holmskov etal, Annu Rev Immunol 21:547-578 (2003); Teh et al., Immunology101:225-232 (2000); Hansen et al, J. Immunol 185(10):6096-6104 (2010).

Ikeda et al. first demonstrated that, like C1q, MBL could activate thecomplement system upon binding to yeast mannan-coated erythrocytes in aC4-dependent manner (Ikeda et al., J. Biol. Chem. 262:7451-7454,(1987)). MBL, a member of the collectin protein family, is acalcium-dependent lectin that binds carbohydrates with 3-and 4-hydroxygroups oriented in the equatorial plane of the pyranose ring. Prominentligands for MBL are thus D-mannose and N-acetyl-D-glucosamine, whilecarbohydrates not fitting this steric requirement have undetectableaffinity for MBL (Weis et al., Nature 360:127-134, (1992)). Theinteraction between MBL and monovalent sugars is extremely weak, withdissociation constants typically in the single-digit millimolar range.MBL achieves tight, specific binding to glycan ligands by avidity, i.e.,by interacting simultaneously with multiple monosaccharide residueslocated in close proximity to each other (Lee et al., Archiv. Biochem.Biophys. 299:129-136, (1992)). MBL recognizes the carbohydrate patternsthat commonly decorate microorganisms such as bacteria, yeast, parasitesand certain viruses. In contrast, MBL does not recognize D-galactose andsialic acid, the penultimate and ultimate sugars that usually decorate“mature” complex glycoconjugates present on mammalian plasma and cellsurface glycoproteins. This binding specificity is thought to promoterecognition of “foreign” surfaces and help protect from“self-activation.” However, MBL does bind with high affinity to clustersof high-mannose “precursor” glycans on N-linked glycoproteins andglycolipids sequestered in the endoplasmic reticulum and Golgi ofmammalian cells (Maynard et al., J. Biol. Chem. 257:3788-3794, (1982)).In addition, it has been shown that MBL can bind the polynucleotides,DNA and RNA, which may be exposed on necrotic and apoptotic cells(Palaniyar et al., Ann. N.Y. Acad. Sci., 1010:467-470 (2003); Nakamuraet al., J. Leuk. Biol. 86:737-748 (2009)). Therefore, damaged cells arepotential targets for lectin pathway activation via MBL binding.

The ficolins possess a different type of lectin domain than MBL, calledthe fibrinogen-like domain. Ficolins bind sugar residues in aCa⁺⁺-independent manner. In humans, three kinds of ficolins (L-ficolin,M-ficolin and H-ficolin) have been identified. The two serum ficolins,L-ficolin and H-ficolin, have in common a specificity forN-acetyl-D-glucosamine; however, H-ficolin also bindsN-acetyl-D-galactosamine. The difference in sugar specificity ofL-ficolin, H-ficolin, CL-11, and MBL means that the different lectinsmay be complementary and target different, though overlapping,glycoconjugates. This concept is supported by the recent report that, ofthe known lectins in the lectin pathway, only L-ficolin bindsspecifically to lipoteichoic acid, a cell wall glycoconjugate found onall Gram-positive bacteria (Lynch et al., J. Immunol. 172:1198-1202,(2004)). In addition to acetylated sugar moieties, the ficolins can alsobind acetylated amino acids and polypeptides (Thomsen et al., Mol.Immunol. 48(4):369-81 (2011)). The collectins (i.e., MBL) and theficolins bear no significant similarity in amino acid sequence. However,the two groups of proteins have similar domain organizations and, likeC1q, assemble into oligomeric structures, which maximize the possibilityof multisite binding.

The serum concentrations of MBL are highly variable in healthypopulations and this is genetically controlled bypolymorphisms/mutations in both the promoter and coding regions of theMBL gene. As an acute phase protein, the expression of MBL is furtherupregulated during inflammation. L-ficolin is present in serum atconcentrations similar to those of MBL. Therefore, the L-ficolin branchof the lectin pathway is potentially comparable to the MBL arm instrength. MBL and ficolins can also function as opsonins, which allowphagocytes to target MBL- and ficolin-decorated surfaces (see Jack etal., J Leukoc Biol., 77(3):328-36 (2004), Matsushita and Fujita,Immunobiology, 205(4-5):490-7 (2002), Aoyagi et al., J Immunol,174(1):418-25(2005). This opsonization requires the interaction of theseproteins with phagocyte receptors (Kuhlman et al., J. Exp. Med.169:1733, (1989); Matsushita et al., J. Biol. Chem. 271:2448-54,(1996)), the identity of which has not been established.

Human MBL forms a specific and high-affinity interaction through itscollagen-like domain with unique C1r/C1s-like serine proteases, termedMBL-associated serine proteases (MASPs). To date, three MASPs have beendescribed. First, a single enzyme “MASP” was identified andcharacterized as the enzyme responsible for the initiation of thecomplement cascade (i.e., cleaving C2 and C4) (Matsushita et al., J ExpMed 176(6):1497-1502 (1992); Ji et al., J. Immunol. 150:571-578,(1993)). It was subsequently determined that the MASP activity was, infact, a mixture of two proteases: MASP-1 and MASP-2 (Thiel et al.,Nature 386:506-510, (1997)). However, it was demonstrated that theMBL-MASP-2 complex alone is sufficient for complement activation(Vorup-Jensen et al., J. Immunol. 165:2093-2100, (2000)). Furthermore,only MASP-2 cleaved C2 and C4 at high rates (Ambrus et al., J. Immunol.170:1374-1382, (2003)). Therefore, MASP-2 is the protease responsiblefor activating C4 and C2 to generate the C3 convertase, C4b2a. This is asignificant difference from the C1 complex of the classical pathway,where the coordinated action of two specific serine proteases (C1r andC1s) leads to the activation of the complement system. In addition, athird novel protease, MASP-3, has been isolated (Dahl, M. R., et al.,Immunity 15:127-35, 2001). MASP-1 and MASP-3 are alternatively splicedproducts of the same gene.

MASPs share identical domain organizations with those of C1r and C1s,the enzymatic components of the C1 complex (Sim et al., Biochem. Soc.Trans. 28:545, (2000)). These domains include an N-terminal C1r/C1s/seaurchin VEGF/bone morphogenic protein (CUB) domain, an epidermal growthfactor-like domain, a second CUB domain, a tandem of complement controlprotein domains, and a serine protease domain. As in the C1 proteases,activation of MASP-2 occurs through cleavage of an Arg-Ile bond adjacentto the serine protease domain, which splits the enzyme intodisulfide-linked A and B chains, the latter consisting of the serineprotease domain.

MBL can also associate with an alternatively spliced form of MASP-2,known as MBL-associated protein of 19 kDa (MAp19) or smallMBL-associated protein (sMAP), which lacks the catalytic activity ofMASP-2. (Stover, J. Immunol. 162:3481-90, (1999); Takahashi et al., Int.Immunol. 11:859-863, (1999)). MAp19 comprises the first two domains ofMASP-2, followed by an extra sequence of four unique amino acids. Thefunction of Map19 is unclear (Degn et al., J Immunol. Methods, 2011).The MASP-1 and MASP-2 genes are located on human chromosomes 3 and 1,respectively (Schwaeble et al., Immunobiology 205:455-466, (2002)).

Several lines of evidence suggest that there are different MBL-MASPcomplexes and a large fraction of the MASPs in serum is not complexedwith MBL (Thiel, et al., J. Immunol. 165:878-887, (2000)). Both H- andL-ficolin bind to all MASPs and activate the lectin complement pathway,as does MBL (Dahl et al., Immunity 15:127-35, (2001); Matsushita et al.,J. Immunol. 168:3502-3506, (2002)). Both the lectin and classicalpathways form a common C3 convertase (C4b2a) and the two pathwaysconverge at this step.

The lectin pathway is widely thought to have a major role in hostdefense against infection in the naive host. Strong evidence for theinvolvement of MBL in host defense comes from analysis of patients withdecreased serum levels of functional MBL (Kilpatrick, Biochim. Biophys.Acta 1572:401-413, (2002)). Such patients display susceptibility torecurrent bacterial and fungal infections. These symptoms are usuallyevident early in life, during an apparent window of vulnerability asmaternally derived antibody titer wanes, but before a full repertoire ofantibody responses develops. This syndrome often results from mutationsat several sites in the collagenous portion of MBL, which interfere withproper formation of MBL oligomers. However, since MBL can function as anopsonin independent of complement, it is not known to what extent theincreased susceptibility to infection is due to impaired complementactivation.

In contrast to the classical and lectin pathways, no initiators of thealternative pathway have previously been found to fulfill therecognition functions that C1q and lectins perform in the other twopathways. Currently it is widely accepted that the alternative pathwayspontaneously undergoes a low level of turnover activation, which can bereadily amplified on foreign or other abnormal surfaces (bacteria,yeast, virally infected cells, or damaged tissue) that lack the propermolecular elements that keep spontaneous complement activation in check.There are four plasma proteins directly involved in the activation ofthe alternative pathway: C3, factors B and D, and properdin.

Although there is extensive evidence implicating both the classical andalternative complement pathways in the pathogenesis of non-infectioushuman diseases, the role of the lectin pathway is just beginning to beevaluated. Recent studies provide evidence that activation of the lectinpathway can be responsible for complement activation and relatedinflammation in ischemia/reperfusion injury. Collard et al. (2000)reported that cultured endothelial cells subjected to oxidative stressbind MBL and show deposition of C3 upon exposure to human serum (Collardet al., Am. J. Pathol. 156:1549-1556, (2000)). In addition, treatment ofhuman sera with blocking anti-MBL monoclonal antibodies inhibited MBLbinding and complement activation. These findings were extended to a ratmodel of myocardial ischemia-reperfusion in which rats treated with ablocking antibody directed against rat MBL showed significantly lessmyocardial damage upon occlusion of a coronary artery than rats treatedwith a control antibody (Jordan et al., Circulation 104:1413-1418,(2001)). The molecular mechanism of MBL binding to the vascularendothelium after oxidative stress is unclear; a recent study suggeststhat activation of the lectin pathway after oxidative stress may bemediated by MBL binding to vascular endothelial cytokeratins, and not toglycoconjugates (Collard et al., Am. J. Pathol. 159:1045-1054, (2001)).Other studies have implicated the classical and alternative pathways inthe pathogenesis of ischemia/reperfusion injury and the role of thelectin pathway in this disease remains controversial (Riedermann, N. C.,et al., Am. J Pathol. 162:363-367, 2003).

Recent studies have shown that MASP-1 and MASP-3 convert the alternativepathway activation enzyme factor D from its zymogen form into itsenzymatically active form (see Takahashi M. et al., J Exp Med207(1):29-37 (2010); Iwaki et al., J Immunol. 187:3751-58 (2011)). Thephysiological importance of this process is underlined by the absence ofalternative pathway functional activity in plasma of MASP-1/3-deficientmice. Proteolytic generation of C3b from native C3 is required for thealternative pathway to function. Since the alternative pathway C3convertase (C3bBb) contains C3b as an essential subunit, the questionregarding the origin of the first C3b via the alternative pathway haspresented a puzzling problem and has stimulated considerable research.

C3 belongs to a family of proteins (along with C4 and a-2 macroglobulin)that contain a rare posttranslational modification known as a thioesterbond. The thioester group is composed of a glutamine whose terminalcarbonyl group forms a covalent thioester linkage with the sulfhydrylgroup of a cysteine three amino acids away. This bond is unstable andthe electrophilic glutamyl-thioester can react with nucleophilicmoieties such as hydroxyl or amino groups and thus form a covalent bondwith other molecules. The thioester bond is reasonably stable whensequestered within a hydrophobic pocket of intact C3. However,proteolytic cleavage of C3 to C3a and C3b results in exposure of thehighly reactive thioester bond on C3b and, following nucleophilic attackby adjacent moieties comprising hydroxyl or amino groups, C3b becomescovalently linked to a target. In addition to its well-documented rolein covalent attachment of C3b to complement targets, the C3 thioester isalso thought to have a pivotal role in triggering the alternativepathway. According to the widely accepted “tick-over theory”, thealternative pathway is initiated by the generation of a fluid-phaseconvertase, iC3Bb, which is formed from C3 with hydrolyzed thioester(iC3; C3(H₂O)) and factor B (Lachmann, P. J., et al., Springer Semin.Immunopathol. 7:143-162, (1984)). The C3b-like C3(H₂O) is generated fromnative C3 by a slow spontaneous hydrolysis of the internal thioester inthe protein (Pangburn, M. K., et al., J. Exp. Med. 154:856-867, 1981).Through the activity of the C₃(H₂JO)Bb convertase, C3b molecules aredeposited on the target surface thereby initiating the alternativepathway.

Prior to the instant discovery described herein, very little was knownabout the initiators of activation of the alternative pathway.Activators were thought to include yeast cell walls (zymosan), many purepolysaccharides, rabbit erythrocytes, certain immunoglobulins, viruses,fungi, bacteria, animal tumor cells, parasites, and damaged cells. Theonly feature common to these activators is the presence of carbohydrate,but the complexity and variety of carbohydrate structures has made itdifficult to establish the shared molecular determinants which arerecognized. It has been widely accepted that alternative pathwayactivation is controlled through the fine balance between inhibitoryregulatory components of this pathway, such as factor H, factor I, DAF,and CR1, and properdin, the latter of which is the only positiveregulator of the alternative pathway (see Schwaeble W. J. and Reid K.B., Immunol Today 20(1):17-21 (1999)).

In addition to the apparently unregulated activation mechanism describedabove, the alternative pathway can also provide a powerful amplificationloop for the lectin/classical pathway C3 convertase (C4b2a) since anyC3b generated can participate with factor B in forming additionalalternative pathway C3 convertase (C3bBb). The alternative pathway C3convertase is stabilized by the binding of properdin. Properdin extendsthe alternative pathway C3 convertase half-life six to ten-fold.Addition of C3b to the alternative pathway C3 convertase leads to theformation of the alternative pathway C5 convertase.

All three pathways (i.e., the classical, lectin and alternative) havebeen thought to converge at C5, which is cleaved to form products withmultiple proinflammatory effects. The converged pathway has beenreferred to as the terminal complement pathway. C5a is the most potentanaphylatoxin, inducing alterations in smooth muscle and vascular tone,as well as vascular permeability. It is also a powerful chemotaxin andactivator of both neutrophils and monocytes. C5a-mediated cellularactivation can significantly amplify inflammatory responses by inducingthe release of multiple additional inflammatory mediators, includingcytokines, hydrolytic enzymes, arachidonic acid metabolites, andreactive oxygen species. C5 cleavage leads to the formation of C5b-9,also known as the membrane attack complex (MAC). There is now strongevidence that sublytic MAC deposition may play an important role ininflammation in addition to its role as a lytic pore-forming complex.

In addition to its essential role in immune defense, the complementsystem contributes to tissue damage in many clinical conditions. Thus,there is a pressing need to develop therapeutically effective complementinhibitors to prevent these adverse effects.

SUMMARY

In one aspect, the present invention provides an isolated monoclonalantibody or antigen-binding fragment thereof thereof that specificallybinds to the serine protease domain of human MASP-3 (amino acid residues450 to 728 of SEQ ID NO:2) with high affinity (having a K_(D) of lessthan 500 pM), wherein the antibody or antigen-binding fragment thereofinhibits alternative pathway complement activation. In some embodiments,antibody or antigen-binding fragment is characterized by at least one ormore of the following properties: (a) inhibits pro-Factor D maturation;(b) does not bind to human MASP-1 (SEQ ID NO:8); (c) inhibits thealternative pathway at a molar ratio of from about 1:1 to about 2.5:1(MASP-3 target to mAb) in a mammalian subject (d) does not inhibit theclassical pathway (e) inhibits of hemolysis and/or opsonization; (f)inhibits of MASP-3 serine protease substrate-specific cleavage; (g)reduces hemolysis or the reduction of C3 cleavage and C3b surfacedeposition; (h) reduces of Factor B and/or Bb deposition on anactivating surface; (i) reduces resting levels (in circulation, andwithout the experimental addition of an activating surface) of activeFactor D relative to pro-Factor D; (j) reduces the level of activeFactor D relative to pro-Factor D in response to an activating surface;(k) reduces the production of resting and surface-induced levels offluid-phase Ba, Bb, C3b, or C3a; and/or (1) reduces factor P deposition.In some embodiments, the isolated antibody or antigen-binding fragmentthereof of paragraph 1 or 2, wherein said antibody or antigen-bindingfragment thereof specifically binds to an epitope located within theserine protease domain of human MASP-3, wherein said epitope is locatedwithin at least one or more of: VLRSQRRDTTVI (SEQ ID NO:9),TAAHVLRSQRRDTTV (SEQ ID NO:10), DFNIQNYNHDIALVQ (SEQ ID NO:11),PHAECKTSYESRS (SEQ ID NO:12), GNYSVTENMFC (SEQ ID NO:13), VSNYVDWVWE(SEQ ID NO:14) and/or VLRSQRRDTTV (SEQ ID NO:15). In some embodiments,the antibody or antigen-binding fragment thereof binds to an epitopewithin at least one of: ECGQPSRSLPSLV (SEQ ID NO:16), RNAEPGLFPWQ (SEQID NO:17); KWFGSGALLSASWIL (SEQ ID NO:18); EHVTVYLGLH (SEQ ID NO:19);PVPLGPHVMP (SEQ ID NO:20); APHMLGL (SEQ ID NO:21); SDVLQYVKLP (SEQ IDNO:22); and/or AFVIFDDLSQRW (SEQ ID NO:23).

In another aspect, the present invention provides an isolated antibody,or antigen-binding fragment thereof, that binds to MASP-3 comprising:(a) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:209 (XXDIN, wherein X at position 1 is S or T and wherein X atposition 2 is N or D); a HC-CDR2 set forth as SEQ ID NO:210(WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8is S, T or R; X at position 9 is I or T; X at position 13 is E or D; Xat position 14 is K or E; and X at position 16 is T or K); and a HC-CDR3set forth as SEQ ID NO:211 (XEDXY, wherein X at position 1 is L or V,and wherein X at position 4 is T or S); and (b) a light chain variableregion comprising a LC-CDR1 set forth as SEQ ID NO:212(KSSQSLLXXRTRKNYLX, wherein X at position 8 is N, I, Q or A; wherein Xat position 9 is S or T; and wherein X at position 17 is A or S); aLC-CDR2 set forth as SEQ ID NO:144 (WASTRES) and a LC-CDR3 set forth asSEQ ID NO:146 (KQSYNLYT).

In another aspect, the present invention provides an isolated antibody,or antigen-binding fragment thereof, that binds to MASP-3 comprising:(a) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:213 (SYGXX, wherein X at position 4 is M or I and wherein X atposition 5 is S or T); a HC-CDR2 set forth as SEQ ID NO:74; and aHC-CDR3 set forth as SEQ ID NO:214 (GGXAXDY, wherein X at position 3 isE or D and wherein X at position 5 is M or L); and (b) a light chainvariable region comprising a LC-CDR1 set forth as SEQ ID NO:215(KSSQSLLDSXXKTYLX , wherein X at position 10 is D, E or A; wherein X atposition 11 is G or A; and wherein X at position 16 is N or S); aLC-CDR2 set forth as SEQ ID NO:155; and a LC-CDR3 set forth as SEQ IDNO:216 (WQGTHFPXT, wherein X at position 8 is W or Y).

In another aspect, the present invention provides an isolated antibody,or antigen-binding fragment thereof, that binds to MASP-3 comprising:(a) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:84 (GKWIE); a HC-CDR2 set forth as SEQ ID NO:86(EILPGTGSTNYNEKFKG) or SEQ ID NO:275 (EILPGTGSTNYAQKFQG); and a HC-CDR3set forth as SEQ ID NO:88 (SEDV); and (b) a light chain variable regioncomprising a LC-CDR1 set forth as SEQ ID NO:142 (KSSQSLLNSRTRKNYLA), SEQID NO:257 (KSSQSLLRTRKNYLA); SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQID NO:259 (KSSQSLLNTRTRKNYLA), a LC-CDR2 set forth as SEQ ID NO:144(WASTRES); and a LC-CDR3 set forth as SEQ ID NO:161 (KQSYNIPT).

In another aspect, the present invention provides an isolated antibody,or antigen-binding fragment thereof, that binds to MASP-3 comprising:(a) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:91 (GYWIE); a HC-CDR2 set forth as SEQ ID NO:93(EMLPGSGSTHYNEKFKG), and a HC-CDR3 set forth as SEQ ID NO:95 (SIDY); and(b) a light chain variable region comprising a LC-CDR1 set forth as SEQID NO:163 (RSSQSLVQSNGNTYLH), a LC-CDR2 set forth as SEQ ID NO:165(KVSNRFS) and a LC-CDR3 set forth as SEQ ID NO:167 (SQSTHVPPT).

In another aspect, the present invention provides an isolated antibody,or antigen-binding fragment thereof, that binds to MASP-3 comprising:

(a) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:109 (RVHFAIRDTNYWMQ), a HC-CDR2 set forth as SEQ ID NO:110(AIYPGNGDTSYNQKFKG), a HC-CDR3 set forth as SEQ ID NO:112 (GSHYFDY); anda light chain variable region comprising a LC-CDR1 set forth as SEQ IDNO:182 (RASQSIGTSIH), a LC-CDR2 set forth as SEQ ID NO:184 (YASESIS) anda LC-CDR3 set forth as SEQ ID NO:186 (QQSNSWPYT); or

(b) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:125 (DYYMN), a HC-CDR2 set forth as SEQ ID NO:127(DVNPNNDGTTYNQKFKG), a HC-CDR3 set forth as SEQ ID NO:129(CPFYYLGKGTHFDY); and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:196 (RASQDISNFLN), a LC-CDR2 set forth as SEQ IDNO:198 (YTSRLHS) and a LC-CDR3 set forth as SEQ ID NO:200 (QQGFTLPWT);or

(c) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:137 a HC-CDR2 set forth as SEQ ID NO:138, a HC-CDR3 set forth asSEQ ID NO:140; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:206, a LC-CDR2 set forth as SEQ ID NO:207 and aLC-CDR3 set forth as SEQ ID NO:208: or

(d) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:98, a HC-CDR2 set forth as SEQ ID NO:99, a HC-CDR3 set forth asSEQ ID NO:101; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:169, a LC-CDR2 set forth as SEQ ID NO:171 and aLC-CDR3 set forth as SEQ ID NO:173; or

(e) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:103, a HC-CDR2 set forth as SEQ ID NO:105, a HC-CDR3 set forth asSEQ ID NO:107; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:176, a LC-CDR2 set forth as SEQ ID NO:178 and aLC-CDR3 set forth as SEQ ID NO:193: or

(f) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:114, a HC-CDR2 set forth as SEQ ID NO:116, a HC-CDR3 set forth asSEQ ID NO:118; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:188, a LC-CDR2 set forth as SEQ ID NO:178 and aLC-CDR3 set forth as SEQ ID NO:190; or

(g) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:114, a HC-CDR2 set forth as SEQ ID NO:121, a HC-CDR3 set forth asSEQ ID NO:123; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:191, a LC-CDR2 set forth as SEQ ID NO:178 and aLC-CDR3 set forth as SEQ ID NO:193.

In another aspect, the present invention provides a method of inhibitingalternative pathway complement activation in a mammal, the methodcomprising administering to a mammal subject in need thereof an amountof a composition comprising a high affinity MASP-3 inhibitory antibodyor antigen-binding fragment thereof sufficient to inhibit alternativepathway complement pathway activation in the mammal. In one embodimentof the method, the antibody, or antigen binding fragment thereof bindsto MASP-3 with an affinity of less than 500 pM. In one embodiment of themethod, as a result of administering the composition comprising theantibody or antigen-binding fragment one or more of the following ispresent in the mammalian subject: (a) inhibition of Factor D maturation;(b) inhibition of the alternative pathway when administered to thesubject at a molar ratio of from about 1:1 to about 2.5:1 (MASP-3 targetto mAb); (c) the classical pathway is not inhibited; (d) inhibition ofhemolysis and/or opsonization; (e) a reduction of hemolysis or thereduction of C3 cleavage and C3b surface deposition; (f) a reduction ofFactor B and Bb deposition on an activating surface; (g) a reduction ofresting levels (in circulation, and without the experimental addition ofan activating surface) of active Factor D relative to pro-Factor D; (h)a reduction of levels of active Factor D relative to pro-Factor D inresponse to an activating surface; and/or (i) a reduction of theproduction of resting and surface-induced levels of fluid-phase Ba, Bb,C3b, or C3a. In one embodiment of the method, the composition comprisesan MASP-3 inhibitory antibody that inhibits the alternative pathway at amolar ratio of from about 1:1 to about 2.5:1 (MASP-3 target to mAb).

In another aspect, the present invention provides a method of inhibitingMASP-3-dependent complement activation in a subject suffering fromparoxysmal nocturnal hemoglobinuria (PNH), age-related maculardegeneration (AMD), ischemia-reperfusion injury, arthritis, disseminatedintravascular coagulation, thrombotic microangiopathy, asthma, densedeposit disease, pauci-immune necrotizing crescentic glomerulonephritis,traumatic brain injury, aspiration pneumonia, endophthalmitis,neuromyelitis optica or Behcet's disease. The method includes the stepof administering to the subject a composition comprising an amount of ahigh affinity MASP-3 inhibitory agent effective to inhibitMASP-3-dependent complement activation. In some embodiments, the methodfurther comprises administering to the subject a composition comprisinga MASP-2 inhibitory agent.

In another aspect, the present invention provides a method ofmanufacturing a medicament for use in inhibiting the effects ofMASP-3-dependent complement activation in living subjects in needthereof, comprising combining a therapeutically effective amount of aMASP-3 inhibitory agent in a pharmaceutical carrier. In someembodiments, the MASP-3 inhibitoyr agent is a high affinity MASP-3inhibitory antibody. In some embodiments, the method in accordance withthis aspect of the invention comprises manufacturing a medicament foruse in inhibiting the effects of MASP-3-dependent complement activationin a subject suffering from, or at risk for developing a disease ordisorder selected from the group consisting of paroxysmal nocturnalhemoglobinuria (PNH), age-related macular degeneration (AMD),ischemia-reperfusion injury, arthritis, disseminated intravascularcoagulation, thrombotic microangiopathy, asthma, dense deposit disease,pauci-immune necrotizing crescentic glomerulonephritis, traumatic braininjury, aspiration pneumonia, endophthalmitis, neuromyelitis optica orBehcet's disease. In some embodiments, the method further comprisescombining a therapeutically effective amount of a MASP-2 inhibitoryagent into or with the medicament comprising the MASP-3 inhibitor.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a physiologically acceptable carrier and a highaffinity MASP-3 inhibitory monoclonal antibody or antigen bindingfragment thereof that binds to human MASP-3 and inhibits alternativepathway complement activation. In one embodiment, said high affinityMASP-3 antibody or antigen binding fragment thereof comprises (a) aheavy chain variable region comprising (i) VHCDR1 comprising SEQ IDNO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii)VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable regioncomprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ IDNO:258, or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO:144 and (iii)VLCDR3 comprising SEQ ID NO:161.

In another aspect, the present invention provides a method for treatinga subject suffering from, or at risk for developing paroxysmal nocturnalhemoglobinuria (PNH), comprising administering to the subject apharmaceutical composition comprising an effective amount of a highaffinity monoclonal antibody or antigen binding fragment thereof thatbinds to human MASP-3 and inhibits alternative pathway complementactivation to treat or reduce the risk of PNH in the subject. In oneembodiment antibody or antigen binding fragment thereof comprises (a) aheavy chain variable region comprising (a) a heavy chain variable regioncomprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprisingSEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88;and (b) a light chain variable region comprising (i) VLCDR1 comprisingSEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258, or SEQ ID NO:259 (ii)VLCDR2 comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ IDNO:161. In some embodiments, the pharmaceutical composition increasesthe survival of red blood cells in the subject suffering from PNH. Insome embodiments, wherein the subject suffering from or at risk fordeveloping PNH exhibits one or more symptoms selected from the groupconsisting of (i) below normal levels of hemoglobin, (ii) below normallevels of platelets; (iii) above normal levels of reticulocytes, and(iv) above normal levels of bilirubin. In some embodiments, thepharmaceutical composition is administered systemically (e.g.,subcutaneously, intra-muscularly, intravenously, intra-arterially or asan inhalant) to a subject suffering from, or at risk for developing PNH.In some embodiments, the subject suffering from or at risk for PNH haspreviously undergone, or is currently undergoing treatment with aterminal complement inhibitor that inhibits cleavage of complementprotein C5. In some embodiments, the method further comprisesadministering to the subject a terminal complement inhibitor thatinhibits cleavage of complement protein C5. In some embodiments, theterminal complement inhibitor is a humanized anti-05 antibody orantigen-binding fragment thereof. In some embodiments, the terminalcomplement inhibitor is eculizumab.

In another aspect, the present invention provides a method for treatinga subject suffering from, or at risk for developing arthritis(inflammatory and non-inflammatory arthritides) comprising administeringto the subject a pharmaceutical composition comprising an effectiveamount of a high affinity monoclonal antibody or antigen bindingfragment thereof that binds to human MASP-3 and inhibits alternativepathway complement activation to treat or reduce the risk of arthritisin the subject. In one embodiment, said antibody or antigen bindingfragment thereof comprises (a) a heavy chain variable region comprising(i)VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) alight chain variable region comprising (i) VLCDR1 comprising SEQ IDNO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ ID NO:161. Insome embodidments, the subject is suffering from arthritis selectedfronm the group consisting of osteoarthritis, rheumatoid arthritis,juvenile rheumatoid arthritis, ankylosing spondylitis, Behcet's disease,infection-related arthritis and psoriatic arthritis. In someembodiments, the pharmaceutical composition is administered systemically(i.e., subcutaneously, intra-muscularly, intravenously, intra-arteriallyor as an inhalant). In some embodiments, the pharmaceutical compositionis administered locally to a joint.

As described herein, the various embodiments of the high affinity MASP-3inhibitory antibodies, optionally in combination with the variousembodiments of the MASP-2 inhibitory agents can be used in thepharmaceutical compositions of the invention.

As described herein, the pharmaceutical compositions of the inventioncan be used in accordance with the methods of the invention.

These and other aspects and embodiments of the herein describedinvention will be evident upon reference to the following detaileddescription and drawings. All of the U.S. patents, U.S. patentapplication publications, U.S. patent applications, foreign patents,foreign patent applications and non-patent publications referred to inthis specification are incorporated herein by reference in theirentirety, as if each was incorporated individually.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 illustrates a new understanding of the lectin and alternativepathways;

FIG. 2 is a schematic diagram adapted from Schwaeble et al., Immunobiol205:455-466 (2002), as modified by Yongqing et al., BBA 1824:253 (2012),illustrating the MASP-1, MASP-3 and MAp44 protein domains and the exonsencoding the same;

FIG. 3 depicts the human MASP-3 amino acid sequence (SEQ ID NO:2) withthe leader sequence shown in underline;

FIG. 4 shows an alignment of full length MASP-3 protein from multiplespecies;

FIG. 5 shows an alignment of the SP domain of MASP-3 protein frommultiple species;

FIG. 6 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of MASP-2 KO and WT mice after administration of an infectivedose of 2.6×10⁷ cfu of N. meningitidis serogroup A Z2491, demonstratingthat MASP-2 deficient mice are protected from N. meningitidis inducedmortality, as described in Example 1;

FIG. 7 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of MASP-2 KO and WT mice after administration of an infectivedose of 6×10⁶ cfu of N. meningitidis serogroup B strain MC58,demonstrating that MASP-2 deficient mice are protected from N.meningitidis induced mortality, as described in Example 1;

FIG. 8 graphically illustrates the log cfu/mL of N. meningitidisserogroup B strain MC58 per mL of blood recovered from MASP-2 KO and WTmice at different time points after i.p. infection with 6×10⁶ cfu of N.meningitidis serogroup B strain MC58 (n=3 at different time points forboth groups of mice), demonstrating that although the MASP-2 KO micewere infected with the same dose of N. meningitidis serogroup B strainMC58 as the WT mice, the MASP-2 KO mice have enhanced clearance ofbacteremia as compared to WT, as described in Example 1;

FIG. 9 graphically illustrates the average illness score of MASP-2 KOand WT mice at 3, 6, 12 and 24 hours after infection with 6×10⁶ cfu ofN. meningitidis serogroup B strain MC58, demonstrating that theMASP-2-deficient mice showed much lower illness scores at 6 hours, 12hours, and 24 hours after infection, as compared to WT mice, asdescribed in Example 1;

FIG. 10 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of mice after administration of an infective dose of 4×10⁶ cfuof N. meningitidis serogroup B strain MC58, followed by administration 3hours post-infection of either inhibitory MASP-2 antibody (1 mg/kg) orcontrol isotype antibody, demonstrating that MASP-2 antibody iseffective to treat and improve survival in subjects infected with N.meningitidis as described in Example 2;

FIG. 11 graphically illustrates the log cfu/mL of viable counts of N.meningitidis serogroup B strain MC58 recovered at different time pointsin the human sera samples shown in TABLE 6 taken at various time pointsafter incubation with N. meningitidis serogroup B strain MC58, asdescribed in Example 3;

FIG. 12 graphically illustrates the log cfu/mL of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in thehuman sera samples shown in TABLE 8, showing that complement-dependentkilling of N. meningitidis in human 20% (v/v) serum is MASP-3 andMBL-dependent, as described in Example 3;

FIG. 13 graphically illustrates the log cfu/mL of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in themouse sera samples shown in TABLE 10, showing that the MASP-2 −/−knockout mouse (referred to as “MASP-2 −/−”) serum has a higher level ofbactericidal activity for N. meningitidis than WT mouse serum, whereasin contrast, the MASP-1/3 −/− mouse serum does not have any bactericidalactivity, as described in Example 3;

FIG. 14 graphically illustrates the kinetics of C3 activation underlectin pathway-specific conditions (1% plasma) in WT, C4−/−,MASP-1/3−/−, Factor B−/− and MASP-2−/− mouse sera, as described inExample 4;

FIG. 15 graphically illustrates the level of alternative pathway-driven(AP-driven) C3b deposition on zymosan-coated microtiter plates under“traditional” alternative pathway-specific (AP-specific) conditions(i.e. BBS/EGTA/Mg⁺⁺ without Ca⁺⁺) as a function of serum concentrationin serum samples obtained from MASP-3-deficient, C4-deficient andMBL-deficient human subjects, as described in Example 4;

FIG. 16 graphically illustrates the level of AP-driven C3b deposition onzymosan-coated microtiter plates under “traditional” AP-specificconditions (i.e., BBS/EGTA/Mg⁻⁺ without Ca⁺⁺) as a function of time in10% human serum samples obtained from MASP-3-deficient, C4-deficient andMBL-deficient human subjects, as described in Example 4;

FIG. 17A graphically illustrates the level of C3b deposition onmannan-coated microtiter plates as a function of serum concentration inserum samples obtained from WT, MASP-2-deficient, and MASP-1/3-deficientmice under “traditional” AP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺without Ca⁺⁺) or under physiological conditions allowing both the lectinpathway and the alternative pathway (AP) to function (BBS/Mg⁺⁺/Ca⁺⁺), asdescribed in Example 4;

FIG. 17B graphically illustrates the level of C3b deposition onzymosan-coated microtiter plates as a function of serum concentration inserum samples obtained from WT, MASP-2-deficient, and MASP-1/3-deficientmice under traditional AP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺without Ca⁺⁺) or under physiological conditions allowing both the lectinpathway and the alternative pathway to function (BBS/Mg⁺⁺/Ca⁺⁺), asdescribed in Example 4;

FIG. 17C graphically illustrates the level of C3b deposition on S.pneumoniae D39-coated microtiter plates as a function of serumconcentration in serum samples obtained from WT, MASP-2-deficient, andMASP-1/3-deficient mice under traditional AP-specific conditions (i.e.BBS/EGTA/Mg⁺⁺ 0 without Ca⁺⁺) or under physiological conditions allowingboth the lectin pathway and the alternative pathway to function(BBS/Mg⁺⁺/Ca⁺⁺), as described in Example 4;

FIG. 18A graphically illustrates the results of a C3b deposition assayin highly diluted sera carried out on mannan-coated microtiter platesunder traditional AP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺ withoutCa⁺⁺) or under physiological conditions allowing both the lectin pathwayand the alternative pathway to function (BBS/Mg⁺⁺/Ca⁺⁺), using serumconcentrations ranging from 0% up to 1.25%, as described in Example 4;

FIG. 18B graphically illustrates the results of a C3b deposition assaycarried out on zymosan-coated microtiter plates under traditionalAP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺ without Ca⁺⁺) or underphysiological conditions allowing both the lectin pathway and thealternative pathway to function (BB S/Mg⁺⁺/Ca⁺⁺), using serumconcentrations ranging from 0% up to 1.25%, as described in Example 4;

FIG. 18C graphically illustrates the results of a C3b deposition assaycarried out on S. pneumoniae D39-coated microtiter plates undertraditional AP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺ without Ca⁺⁺) orunder physiological conditions allowing both the lectin pathway and thealternative pathway to function (BBS/Mg⁺⁺/Ca⁺⁺), using serumconcentrations ranging from 0% up to 1.25%, as described in Example 4;

FIG. 19 graphically illustrates the level of hemolysis (as measured byhemoglobin release of lysed mouse erythrocytes (Crry/C3−/−) into thesupernatant measured by photometry) of mannan-coated murine erythrocytesby human serum under physiological conditions (i.e., in the presence ofCa⁺⁺) over a range of serum dilutions in serum from MASP-3−/−, heatinactivated normal human serum (HI NHS), MBL−/−, NHS +MASP-2 monoclonalantibody and NHS control, as described in Example 5;

FIG. 20 graphically illustrates the level of hemolysis (as measured byhemoglobin release of lysed mouse erythrocytes (Crry/C3−/−) into thesupernatant measured by photometry) of mannan-coated murine erythrocytesby human serum under physiological conditions (i.e., in the presence ofCa⁺⁺) over a range of serum concentration in serum from MASP-3−/−, heatinactivated (HI) NHS, MBL−/−, NHS +MASP-2 monoclonal antibody and NHScontrol, as described in Example 5;

FIG. 21 graphically illustrates the level of hemolysis (as measured byhemoglobin release of lysed WT mouse erythrocytes into the supernatantmeasured by photometry) of non-coated murine erythrocytes by human serumunder physiological conditions (i.e., in the presence of Ca⁺⁺) over arange of serum concentrations in serum from 3MC (MASP-3−/−), heatinactivated (HI) NHS, MBL−/−, NHS+MASP-2 monoclonal antibody and NHScontrol, as described in Example 5;

FIG. 22 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed mouse erythrocytes (CD55/59−/−) into the supernatantmeasured by photometry) of non-coated murine erythrocytes by human serumunder physiological conditions (i.e., in the presence of Ca⁺⁺) over arange of serum concentrations in serum from heat inactivated (HI) NHS,MBL−/−, NHS+MASP-2 monoclonal antibody and NHS control, as described inExample 5;

FIG. 23 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed rabbit erythrocytes into the supernatant measured byphotometry) of mannan-coated rabbit erythrocytes by MASP-1/3−/− mouseserum and WT control mouse serum under physiological conditions (i.e.,in the presence of Ca⁺⁺) over a range of serum concentrations, asdescribed in Example 6;

FIG. 24A is a FACS histogram of MASP-3 antigen/antibody binding forclone M3J5, as described in Example 7;

FIG. 24B is a FACS histogram of MASP-3 antigen/antibody binding forclone M3M1, as described in Example 7;

FIG. 25 graphically illustrates a saturation binding curve of clone M3J5(Clone 5) for the MASP-3 antigen, as described in Example 7;

FIG. 26A is an amino acid sequence alignment of the VH regions of M3J5,M3M1, D14 and 1E10 to the chicken DT40 VH sequence, wherein dotsrepresent amino acid identity with the DT40 sequence and dashes indicatespaces introduced to maximize the alignment, as described in Example 7;

FIG. 26B is an amino acid sequence alignment of the VL regions of M3J5,M3M1, D14 and 1E10 to the chicken DT40 VL sequence, wherein dotsrepresent amino acid identity with the DT40 sequence and dashes indicatespaces introduced to maximize the alignment, as described in Example 7;

FIG. 27 is a bar graph showing the inhibitory activity of the monoclonalantibody (mAb) 1E10 in the Wieslab Complement System Screen, MBL Pathwayin comparison to the positive serum provided with the assay kit, as wellas an isotype control antibody, demonstrating that mAb1E10 partialinhibits LEA-2-dependent activation, (via inhibition of MASP-1-dependentactivation of MASP-2), whereas the isotype control antibody does not, asdescribed in Example 7;

FIG. 28A provides the results of flow cytometry analysis for C3bdeposition on heat-killed Staphylococcus aureus demonstrating that innormal human serum in the presence of EDTA, which is known to inactivatethe lectin and alternative pathways, no C3b deposition was observed(panel 1), in normal human serum treated with Mg⁺⁺/EGTA, alternativepathway-driven C3b deposition is observed (panel 2), and as shown inpanel 3, 4 and 5, in factor B-depleted, factor D-depleted and properdin(factor P)-depleted serum, respectively, no alternative pathway drivenC3b deposition is observed, as described in Example 8;

FIG. 28B provides the results of flow cytometry analysis for C3bdeposition on heat-killed S. aureus demonstrating that, as inEDTA-treated normal serum (panel 1), AP-driven C3b deposition is absentin 3MC serum in the presence of Mg⁺⁺/EGTA (panel 3), whereas panels 4and 5 show that active full length rMASP-3 (panel 4) and active rMASP-3(CCP1-CCP2-SP) (panel 5) both restore AP-driven C3b deposition in 3MCserum to levels observed in normal serum treated with Mg⁺⁺/EGTA (panel2), neither inactive rMASP-3 (S679A) (panel 6) nor wild type rMASP-1(panel 7) can restore AP-driven C3b deposition in 3MC serum, asdescribed in Example 8;

FIG. 29 shows the results of a Western blot analysis to determine factorB cleavage in response to S. aureus in 3MC serum in the presence orabsence of rMASP-3, demonstrating that the normal human serum in thepresence of EDTA (negative control, lane 1) demonstrates very littleFactor B cleavage relative to normal human serum in the presence ofMg⁺⁺/EGTA, shown in lane 2 (positive control), as further shown in lane3, 3MC serum demonstrates very little Factor B cleavage in the presenceof Mg⁺⁺/EGTA. However, as shown in lane 4, Factor B cleavage is restoredby the addition and pre-incubation of full-length, recombinant MASP-3protein to the 3MC serum, as described in Example 8;

FIG. 30 shows Comassie staining of a protein gel in which Factor Bcleavage is analyzed, demonstrating that Factor B cleavage is mostoptimal in the presence of C3, MASP-3 and pro-factor D (lane 1), and asshown in lanes 4 and 5, either MASP-3 or pro-factor D alone are able tomediate Factor B cleavage, as long as C3 is present, as described inExample 8;

FIG. 31 graphically illustrates the mean fluorescent intensities (MFI)of C3b staining of S. aureus obtained from mAbD14 (which binds MASP-3),mAb1A5 (negative control antibody) and an isotype control antibodyplotted as a function of mAb concentration in 3MC serum in the presenceof rMASP-3, demonstrating that mAbD14 inhibits MASP-3-dependent C3bdeposition in a concentration-dependent manner, as described in Example8;

FIG. 32 shows Western blot analysis of pro-factor D substrate cleavage,wherein compared to pro-factor D alone (lane 1) or the inactive fulllength recombinant MASP-3 (S679A; lane 3) or MASP-1 (S646A; lane 4),full length wild type recombinant MASP-3 (1ane2) and MASP-1 (lane 5)either completely or partially cleave pro-factor D to generate maturefactor D, as described in Example 9;

FIG. 33 is a Western blot showing the inhibitory activity of the MASP-3binding mAbs D14 (lane 2) and M3M1 (lane 3) on MASP-3-dependentpro-factor D cleavage in comparison to a control reaction containingonly MASP-3 and pro-factor D (no mAb, lane 1), as well as a controlreaction containing a mAb obtained from the DTLacO library that bindsMASP-1, but not MASP-3 (lane 4), as described in Example 9;

FIG. 34 graphically illustrates the level of AP-driven C3b deposition onzymosan-coated microtiter plates as a function of serum concentration inserum samples obtained from MASP-3-deficient (3MC), C4-deficient andMBL-deficient subjects, demonstrating that MASP-3-deficient sera fromPatient 2 and Patient 3 have residual AP activity at high serumconcentrations (25%, 12.5%, 6.25% serum concentrations), but asignificantly higher AP₅₀ (i.e., 8.2% and 12.3% of serum needed toachieve 50% of maximum C3 deposition), as described in Example 10;

FIG. 35A graphically illustrates the level of AP-driven C3b depositionon zymosan-coated microtiter plates under “traditional” AP-specificconditions (i.e., BBS/EGTA/Mg⁻⁺ without Ca⁺⁺) as a function of time in10% human serum samples obtained from MASP-3 deficient, C4-deficient andMBL-deficient human subjects, as described in Example 10;

FIG. 35B shows a western blot with plasma obtained from 3MC patient #2(MASP-3 (−/−), MASP-1 (+/+)), 3MC patient #3 (MASP-3 (−/−), MASP-1(−/−)), and sera from normal donors (W), wherein human pro-factor D(25,040 Da) and/or mature factor D (24,405 Da) was detected with a humanfactor D-specific antibody, as described in Example 10;

FIG. 35C graphically illustrates the results of the Weislab classical,lectin and alternative pathway assays with plasma obtained from 3MCpatient #2, 3MC patient #3, and normal human serum, as described inExample 10;

FIG. 36 graphically illustrates the percent hemolysis (as measured byhemoglobin release of lysed rabbit erythrocytes into the supernatantmeasured by photometry) of mannan-coated rabbit erythrocytes over arange of serum concentrations in serum from two normal human subjects(NHS) and from two 3MC patients (Patient 2 and Patient 3), measured inthe absence of Ca⁺⁺, demonstrating that MASP-3 deficiency reduces thepercentage of complement-mediated lysis of mannan-coated erythrocytes ascompared to normal human serum, as described in Example 10;

FIG. 37 graphically illustrates the level of AP-driven C3b deposition onzymosan-coated microtiter plates as a function of the concentration ofrecombinant full length MASP-3 protein added to serum samples obtainedfrom human 3MC Patient 2 (MASP-3^(−/−)), demonstrating that, compared tothe negative control inactive recombinant MASP-3 (MASP-3A; S679A),active recombinant MASP-3 protein reconstitutes AP-driven C3b depositionon zymosan-coated plates in a concentration-dependent manner, asdescribed in Example 10;

FIG. 38 graphically illustrates the percent hemolysis (as measured byhemoglobin release of lysed rabbit erythrocytes into the supernatantmeasured by photometry) of mannan-coated rabbit erythrocytes over arange of serum concentrations in (1) normal human serum (NETS); (2) 3MCpatient serum; (3) 3MC patient serum plus active full length recombinantMASP-3 (20 μg/ml); and (4) heat-inactivated human serum (HIS), measuredin the absence of Ca⁺⁺, demonstrating that the percent lysis of rabbiterythrocytes is significantly increased in 3MC serum containing rMASP-3as compared to the percent lysis in 3MC serum without recombinant MASP-3(p=0.0006), as described in Example 10;

FIG. 39 graphically illustrates the percentage of rabbit erythrocytelysis in 7% human serum from 3MC Patient 2 and from 3MC Patient 3containing active recombinant MASP-3 at a concentration range of 0 to110 μg/ml (in BBS/Mg⁺⁺/EGTA, demonstrating that the percentage of rabbiterythrocyte lysis increases with the amount of recombinant MASP-3 in aconcentration-dependent manner, as described in Example 10;

FIG. 40 graphically illustrates the level of LEA-2-driven C3b depositionon Mannan-coated ELISA plates as a function of the concentration ofhuman serum diluted in BBS buffer, for serum from a normal human subject(NHS), from two 3MC patients (Patient 2 and Patient 3), from the parentsof Patient 3 and from a MBL-deficient subject, as described in Example10;

FIG. 41 graphically illustrates a representative example of a bindingexperiment that was performed with human MASP-3 in which the M3-1 Fab(also referred to as 13B1) shows an apparent binding affinity (EC₅₀) ofabout 0.117 nM to the human protein, as described in Example 11;

FIG. 42 graphically illustrates a representative example of a bindingexperiment that was performed with mouse MASP-3 in which the M3-1 Fab(also referred to as 13B1) shows an apparent binding affinity (EC₅₀) ofabout 0.214 nM to the mouse protein, as described in Example 11;

FIG. 43 graphically illustrates the level of complement factor Bbdeposition on zymosan particles (determined by cytometric detectionmeasured in MFI units) in the presence of varying concentrations of mAbM3-1 (also referred to as 13B1) in CFD-depleted human serum, asdescribed in Example 11;

FIG. 44 graphically illustrates the level of C3 deposition on zymosanparticles at various time points after a single dose of mAb M3-1 (13B1)(10 mg/kg i.v.) in wild-type mice, as described in Example 11;

FIG. 45 graphically illustrates the percent survival of donor RBCs (WTor Crry−) over a period of 14 days in wild-type recipient mice treatedwith mAb M3-1 (13B1) (10 mg/kg on days −11, 04, −1 and +6), mAb BB5.1treated, or vehicle treated mice, as described in Example 12;

FIG. 46 graphically illustrates the percent survival of donor RBCs (WTor Crry−) over a period of 16 days in wild-type recipient mice treatedwith a single dose of mAb M3-1 (13B1) (20 mg/kg on day −6) or vehicletreated mice, as described in Example 12;

FIG. 47 graphically illustrates the clinical scores of the mice treatedwith mAb M3-1 (13B1) (5 mg/kg or 20 mg/kg) or vehicle treated mice overa 14 day time course in a collagen-antibody induced arthritis model, asdescribed in Example 13;

FIG. 48 graphically illustrates the percent incidence of arthritis ofthe mice treated with mAb M3-1 (13B1) (5 mg/kg or 20 mg/kg) or vehicletreated mice over a 14 day time course in a collagen-antibody inducedarthritis model, as described in Example 13;

FIG. 49A shows the amino acid sequences of the VH regions of highaffinity (≤500 pM) anti-human MASP-3 inhibitory mAbs, as described inExample 15;

FIG. 49B shows the amino acid sequences of the VL regions of highaffinity (≤500 pM) anti-human MASP-3 inhibitory mAbs, as described inExample 15;

FIG. 50A is a dendrogram of the VH regions of high affinity anti-humanMASP-3 inhibitory mAbs, as described in Example 15;

FIG. 50B is a dendrogram of the VL regions of high affinity anti-humanMASP-3 inhibitory mAbs, as described in Example 15;

FIG. 51A graphically illustrates the results of a binding experiment inwhich representative purified recombinant anti-human MASP-3 inhibitoryantibodies show an apparent binding avidity of less than 500 pM (e.g.,from 240 pM to 23 pM) to the human MASP-3 protein, as described inExample 16;

FIG. 51B graphically illustrates the results of a binding experiment inwhich representative purified recombinant anti-human MASP-3 inhibitoryantibodies show an apparent binding avidity of less than 500 pM (e.g.,from 91 pM to 58 pM) to the human MASP-3 protein, as described inExample 16;

FIG. 51C graphically illustrates the results of a binding experiment inwhich representative purified recombinant high affinity anti-humanMASP-3 inhibitory antibodies are shown to be selective for binding toMASP-3 and do not bind to human MASP-1, as described in Example 16;

FIG. 51D graphically illustrates the results of a binding experiment inwhich representative purified recombinant high affinity anti-humanMASP-3 inhibitory antibodies are shown to be selective for binding toMASP-3 and do not bind to human MASP-2, as described in Example 16;

FIG. 52 graphically illustrates the results of a binding experiment inwhich representative purified recombinant anti-human MASP-3 inhibitoryantibodies also show high binding avidity to the mouse MASP-3 protein,as described in Example 16;

FIG. 53 graphically illustrates the results of an experiment measuringthe ability of representative high affinity MASP-3 antibodies to inhibitfluorogenic tripeptide cleavage, as described in Example 16;

FIG. 54 shows a Western blot demonstrating the ability of representativehigh affinity MASP-3 inhibitory mAbs to block recombinantMASP-3-mediated cleavage of pro-factor D to factor D in an in vitroassay, as described in Example 16;

FIG. 55A graphically illustrates the level of complement factor Bbdeposition on zymosan particles (determined by flow cytometric detectionmeasured in MFI units) in the presence of varying concentrations of highaffinity MASP-3 mAbs 1F3, 1G4, 2D7 and 4B6 in factor D-depleted humanserum, as described in Example 16;

FIG. 55B graphically illustrates the level of complement factor Bbdeposition on zymosan particles (determined by flow cytometric detectionmeasured in MFI units) in the presence of varying concentrations of highaffinity MASP-3 mAbs 4D5, 10D12 and 13B1 in factor D-depleted humanserum, as described in Example 16;

FIG. 56A graphically illustrates the level of inhibition of rabbiterythrocyte lysis in the presence of varying concentrations of highaffinity MASP-3 mAbs 1A10, 1F3, 4B6, 4D5and 2F2 as described in Example16;

FIG. 56B graphically illustrates the level of inhibition of rabbiterythrocyte lysis in the presence of varying concentrations of highaffinity MASP-3 mAbs 1B11, 1E7, 1G4, 2D7 and 2F5 as described in Example16;

FIG. 57 shows a Western blot analyzing the level of pro-Factor D) andFactor D in 3MC patient serum (Patient B) in the presence of activerecombinant MASP-3 (rMASP-3), inactive rMASP-3, and active rMASP-3 plushigh affinity MASP-3 mAb 4D5, as described in Example 16;

FIG. 58 graphically illustrates the level of C3/C3b/iC3b deposition onzymosan particles at various time points after a single dose of highaffinity MASP-3 mAbs M3-1 (13B1, 10 mg/kg) or 10D12 (10 mg/kg) inwild-type mice, as described in Example 17;

FIG. 59 shows a Western blot analyzing the status of the Factor Bafragment of Factor B in mice treated with high affinity MASP-3 mAb 10D12(10 mg/kg) or vehicle control treated mice, as described in Example 17;

FIG. 60 graphically illustrates the level of inhibition of hemolysis by20% serum from mice treated with high affinity MASP-3 mAb 10D12 (10mg/kg or 25 mg/kg), as described in Example 17;

FIG. 61A graphically illustrates the results of competition bindinganalysis to identify high affinity MASP-3 mAbs that block theinteraction between high affinity MASP-3 mAb 4D5 and human MASP-3, asdescribed in Example 18;

FIG. 61B graphically illustrates the results of competition bindinganalysis to identify high affinity MASP-3 mAbs that block theinteraction between high affinity MASP-3 mAb 10D12 and human MASP-3, asdescribed in Example 18;

FIG. 61C graphically illustrates the results of competition bindinganalysis to identify high affinity MASP-3 mAbs that block theinteraction between high affinity MASP-3 mAb 13B1 and human MASP-3, asdescribed in Example 18;

FIG. 61D graphically illustrates the results of competition bindinganalysis to identify high affinity MASP-3 mAbs that block theinteraction between high affinity MASP-3 mAb 1F3 and human MASP-3, asdescribed in Example 18;

FIG. 61E graphically illustrates the results of competition bindinganalysis to identify high affinity MASP-3 mAbs that block theinteraction between high affinity MASP-3 mAb 1G4 and human MASP-3, asdescribed in Example 18;

FIG. 62 provides a schematic diagram showing the regions of contact onhuman MASP-3 by the high affinity MASP-3 mAbs, as determined by Pepscananalysis, as described in Example 18;

FIG. 63A shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAbs 1F3, 4D5 and 1A10, including amino acid residues498-509 (SEQ ID NO:9), amino acid residues 544-558 (SEQ ID NO:11), aminoacid residues 639 to 649 (SEQ ID NO:13) and amino acid residues 704 to713 (SEQ ID NO:14) of MASP-3, as described in Example 18;

FIG. 63B shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAb 10D12, including amino acid residues 498 to 509 (SEQID NO:9) of MASP-3, as described in Example 18;

FIG. 64 shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAb 13B1, including amino acid residues 494 to 508 (SEQID NO:10) and amino acid residues 626 to 638 (SEQ ID NO: 12) of MASP-3,as described in Example 18;

FIG. 65 shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAb 1B11, including amino acid residues 435 to 447 (SEQID NO:16), amino acid residues 454 to 464 (SEQ ID NO:17), amino acidresidues 583 to 589 (SEQ ID NO:21) and amino acid residues 614 to 623(SEQ ID NO:22) of MASP-3, as described in Example 18;

FIG. 66 shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAbs 1E7, 1G4 and 2D7, including amino acid residues 454to 464 (SEQ ID NO:17), amino acid residues 514 to 523 (SEQ ID NO:19) andamino acid residues 667 to 678 (SEQ ID NO:23) of MASP-3, as described inExample 18;

FIG. 67 shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAbs 15D9 and 2F5, including amino acid residues 454 to464 (SEQ ID NO:17), amino acid residues 479 to 493 (SEQ ID NO:18), aminoacid residues 562 to 571 (SEQ ID NO:20), and amino acid residues 667 to678 (SEQ ID NO:23) of MASP-3, as described in Example 18;

FIG. 68 graphically illustrates the results of the Experimentalautoimmune encephalomyelitis (EAE) model in mice treated with eitherhigh affinity MASP-3 inhibitory mAb 13B1 (10 mg/kg), Factor B mAb 1379(30 mg/kg) or isotype control mAb (10 mg/kg), as described in Example20;

FIG. 69 graphically illustrates APC activity, as determined by theaverage MFI in a flow cytometric assay detecting complement factor Bb onthe surface of zymosan particles, in serum samples obtained from a groupof three cynomolgus monkeys over time after treatment with high affinityMASP-3 mAb h13B1X, either in the presence or absence of anti-factor Dantibody spiked into the serum sample, as described in Example 21;

FIG. 70 graphically illustrates APC activity, as determined by Bbdeposition on zymosan, in serum samples obtained from groups ofcynomolgus monkeys (3 animals per group) treated with a single 5 mg/kgintravenous dose of high affinity MASP-3 inhibitory mAbs h4D5X, h10D12Xor h13B1X, as described in Example 21;

FIG. 71A graphically illustrates APC activity, as determined byfluid-phase Ba in serum samples obtained from groups of cynomolgusmonkeys (3 animals per group) over time after treatment with a single 5mg/kg intravenous dose of mAbs h4D5X, h10D12X, and h13B1X, as describedin Example 21;

FIG. 71B graphically illustrates APC activity, as determined byfluid-phase Bb in serum samples obtained from groups of cynomolgusmonkeys (3 animals per group) over time after treatment with a single 5mg/kg intravenous dose of mAbs h4D5X, h10D12X, and h13B1X, as describedin Example 21;

FIG. 71C graphically illustrates APC activity, as determined byfluid-phase C3a in serum samples obtained from groups of cynomolgusmonkeys (3 animals per group) over time after treatment with a single 5mg/kg intravenous dose of mAbs h4D5X, h10D12X, and h13B1X, as describedin Example 21;

FIG. 72A graphically illustrates the molar ratio of target (MASP-3) tothe high affinity MASP-3 inhibitory antibody h4D5X at the timepoints ofcomplete APC inhibition, as measured by fluid-phase Ba, as described inExample 21;

FIG. 72B graphically illustrates the molar ratio of target (MASP-3) tothe high affinity MASP-3 inhibitory antibody h10D12X at the timepointsof complete APC inhibition, as measured by fluid-phase Ba, as describedin Example 21;

FIG. 72C graphically illustrates the molar ratio of target (MASP-3) tothe high affinity MASP-3 inhibitory antibody h13B1X at the timepoints ofcomplete APC inhibition, as measured by fluid-phase Ba, as described inExample 21; and

FIG. 73 shows a Western blot analyzing the level of pro-Factor D andFactor D in serum from a cynomolgus monkey over time (hours) aftertreatment with a single 5 mg/kg intravenous dose of mAb h13B1X, asdescribed in Example 21.

DESCRIPTION OF SEQUENCE LISTING

-   SEQ ID NO:1 human MASP-3 cDNA-   SEQ ID NO:2 human MASP-3 protein (with leader)-   SEQ ID NO:3 mouse MASP-3 protein (with leader)-   SEQ ID NO:4 rat MASP-3 protein-   SEQ ID NO:5 chicken MASP-3 protein-   SEQ ID NO:6 rabbit MASP-3 protein-   SEQ ID NO:7 Cynomolgus monkey MASP-3 protein-   SEQ ID NO:8 human MASP-1 protein (with leader)

Human MASP-3 SP domain peptide fragments:

-   SEQ ID NO:9 (aa 498-509 of human MASP-3 w/leader)-   SEQ ID NO:10 (aa 494-508 of human MASP-3 w/leader)-   SEQ ID NO:11 (aa 544-558 of human MASP-3 w/leader)-   SEQ ID NO:12 (aa 626-638 of human MASP-3 w/leader)-   SEQ ID NO:13 (aa 639-649 of human MASP-3 w/leader)-   SEQ ID NO:14 (aa 704-713 of human MASP-3 w/leader)-   SEQ ID NO:15 (aa 498-508 of human MASP-3 w/leader)-   SEQ ID NO:16 (aa 435-447 of human MASP-3 w/leader)-   SEQ ID NO:17 (aa 454-464 of human MASP-3 w/leader)-   SEQ ID NO:18 (aa 479-493 of human MASP-3 w/leader)-   SEQ ID NO:19 (aa 514-523 of human MASP-3 w/leader)-   SEQ ID NO:20 (aa 562-571 of human MASP-3 w/leader)-   SEQ ID NO:21 (aa 583-589 of human MASP-3 w/leader)-   SEQ ID NO:22 (aa 614-623 of human MASP-3 w/leader)-   SEQ ID NO:23 (aa 667-678 of human MASP-3 w/leader)-   SEQ ID NO:24-39: Heavy chain variable regions-mouse parental-   SEQ ID NO:24 4D5_VH-   SEQ ID NO:25 1F3_VH-   SEQ ID NO:26 4B6_VH-   SEQ ID NO:27 1A10_VH-   SEQ ID NO:28 10D12_VH-   SEQ ID NO:29 35C1_VH-   SEQ ID NO:30 13B1_VH-   SEQ ID NO:31 1G4_VH-   SEQ ID NO:32 1E7_VH-   SEQ ID NO:33 2D7_VH-   SEQ ID NO:34 49C11_VH-   SEQ ID NO:35 15D9_VH-   SEQ ID NO:36 2F5_VH-   SEQ ID NO:37 1B11_VH-   SEQ ID NO:38 2F2_VH-   SEQ ID NO:39 11B6_VH-   SEQ ID NO:40-54: Light chain variable regions-mouse parental-   SEQ ID NO:40 4D5_VL-   SEQ ID NO:41 1F3_VL-   SEQ ID NO:42 4B6/1A10_VL-   SEQ ID NO:43 10D12_VL-   SEQ ID NO:44 35C1_VL-   SEQ ID NO:45 13B1_VL-   SEQ ID NO:46 1G4_VL-   SEQ ID NO:47 1E7_VL-   SEQ ID NO:48 2D7_VL-   SEQ ID NO:49 49C11_VL-   SEQ ID NO:50 15D9_VL-   SEQ ID NO:51 2F5_VL-   SEQ ID NO: 52 1B11_VL-   SEQ ID NO:53 2F2_VL-   SEQ ID NO:54 11B6_VL-   SEQ ID NO:55-140: heavy chain framework regions (FR) and    complementarity-determining regions (CDRs) from mouse parental    MASP-3 mAbs-   SEQ ID NO:141-208: light chain I/R and CDRs from mouse parental    MASP-3 mAbs-   SEQ ID NO:209-216: CDR consensus sequences-   SEQ ID NO:217-232: DNA encoding heavy chain variable regions (mouse    parental)-   SEQ ID NO:233-247: DNA encoding light chain variable regions (mouse    parental)-   SEQ ID NO:248: humanized 4D5_VH-14 (h4D5_VH-14) heavy chain variable    region-   SEQ ID NO:249: humanized 4D5_VH-19 (h4D5_VH-19) heavy chain variable    region-   SEQ ID NO:250: humanized 4D5_VL-1 (h4D5 VL-1) light chain variable    region-   SEQ ID NO:251: humanized 10D12_VH-45 (h10D12_VH-45) heavy chain    variable region-   SEQ ID NO:252: humanized 10D12_VH-49 (h10D12_VH-49) heavy chain    variable region-   SEQ ID NO:253: humanized 10D12_VL-21 (h10D12-VL-21) light chain    variable region-   SEQ ID NO:254: humanized 13B1_VH-9 (h13B1-VH-9) heavy chain variable    region-   SEQ ID NO:255: humanized 13B1_VH-10 (h13B1-VH-10) heavy chain    variable region-   SEQ ID NO:256: humanized 13B1-VL-1 (h13B1-VL-1) light chain variable    region-   SEQ ID NO:257: 4D5 and 13B1 LC-CDR1-NQ-   SEQ ID NO:258: 4D5 and 13B1 LC-CDR1-NA-   SEQ ID NO:259: 4D5 and 13B1 LC-CDR1-ST-   SEQ ID NO:260: consensus LC-CDR1 for 4D5, 13B1 parental and variants-   SEQ ID NO:261: 10D12 LC-CDR1-DE-   SEQ ID NO:262: 10D12 LC-CDR1-DA-   SEQ ID NO:263: 10D12 LC-CDR1-GA-   SEQ ID NO:264-277: HC I/R and CDRs for humanized 4D5, 10D12 and 13B1-   SEQ ID NO:278: h4D5_VL-1-NA-   SEQ ID NO:279: h10D12_VL-21-GA-   SEQ ID NO:280: h13B1_VL-1-NA-   SEQ ID NO:281-287 LC I/R and CDRs for humanized 4D5, 10D12 and 13B1-   SEQ ID NO:288-293: DNA encoding humanized 4D5, 10D12, 13B1 heavy    chain variable region and variants-   SEQ ID NO:294-299: DNA encoding humanized 4D5, 10D12, 13B1 light    chain variable region and variants-   SEQ ID NO:300: parent DTLacO heavy chain variable region (VH)    polypeptide-   SEQ ID NO:301: MASP-3 specific clone M3J5 heavy chain variable    region (VH) polypeptide-   SEQ ID NO:302: MASP-3 specific clone M3M1 heavy chain variable    region (VH) polypeptide-   SEQ ID NO:303: parent DTLacO light chain variable region (VL)    polypeptide-   SEQ ID NO:304: MASP-3 specific clone M3J5 light chain variable    region (VL) polypeptide-   SEQ ID NO:305: MASP-3 specific clone M3M1 light chain variable    region (VL) polypeptide-   SEQ ID NO:306: MASP-3 clone D14 heavy chain variable region (VH)    polypeptide-   SEQ ID NO:307: MASP-3 clone D14 light chain variable region (VL)    polypeptide-   SEQ ID NO:308: MASP-1 clone 1E10 heavy chain variable region (VH)    polypeptide-   SEQ ID NO:309: MASP-1 clone 1E10 light chain variable region (VL)    polypeptide-   SEQ ID NO:310: human IgG4 constant region-   SEQ ID NO:311: human IgG4 constant region with S228P mutation-   SEQ ID NO:312: human IgG4 constant region with S228P mutation_X-   SEQ ID NO:313: human IgK constant region

DETAILED DESCRIPTION I. DEFINITIONS

Unless specifically defined herein, all terms used herein have the samemeaning as would be understood by those of ordinary skill in the art ofthe present invention. The following definitions are provided in orderto provide clarity with respect to the terms as they are used in thespecification and claims to describe the present invention.

As used herein, the lectin pathway effector arm 1 (“LEA-1”) refers tolectin-dependent activation of factor B and factor D by MASP-3.

As used herein, the lectin pathway effector arm 2 (“LEA-2”) refers toMASP-2-dependent complement activation.

As used herein, the term “MASP-3-dependent complement activation”comprises two components: (i) lectin MASP-3-dependent activation offactor B and factor D, encompassed in LEA-1-mediated complementactivation, occurs in the presence of Ca⁺⁺, commonly leading to theconversion of C3bB to C3bBb and of pro-factor D to factor D; and (ii)lectin-independent conversion of factor B and factor D, which can occurin the absence of Ca⁺⁺, commonly leading to the conversion of C3bB toC3bBb and of pro-factor D to factor D. LEA-1-mediated complementactivation and lectin-independent conversion of factor B and factor Dhave been determined to cause opsonization and/or lysis. While notwishing to be bound by any particular theory, it is believed that onlywhen multiple C3b molecules associate and bind in close proximity, theC3bBb C3 convertase changes its substrate specificity and cleaves C5 asthe alternative pathway C5 convertase termed C3bBb(C3b)n.

As used herein, the term “MASP-2-dependent complement activation”, alsoreferred to herein as LEA-2-mediated complement activation, comprisesMASP-2 lectin-dependent activation, which occurs in the presence ofCa⁺⁺, leading to the formation of the lectin pathway C3 convertase C4b2aand upon accumulation of the C3 cleavage product C3b subsequently to theC5 convertase C4b2a(C3b)n, which has been determined to causeopsonization and/or lysis.

As used herein, the term “traditional understanding of the alternativepathway” also referred to as the “traditional alternative pathway”refers to the alternative pathway prior to the instant discoverydescribed herein, i.e., complement activation that is triggered, forexample, by zymosan from fungal and yeast cell walls, lipopolysaccharide(LPS) from Gram negative outer membranes, and rabbit erythrocytes, aswell as from many pure polysaccharides, viruses, bacteria, animal tumorcells, parasites and damaged cells, and which has traditionally beenthought to arise from spontaneous proteolytic generation of C3b fromcomplement factor C3. As used herein, activation of the “traditionalalternative pathway”, also referred to herein as the “alternativepathway”, is measured in Mg⁺⁺/EGTA buffer (i.e., in the absence ofCa⁺⁺).

As used herein, the term “lectin pathway” refers to complementactivation that occurs via the specific binding of serum and non-serumcarbohydrate-binding proteins including mannan-binding lectin (MBL),CL-11 and the ficolins (H-ficolin, M-ficolin, or L-ficolin). Asdescribed herein, the inventors have discovered that the lectin pathwayis driven by the two effector arms, lectin pathway effector arm 1(LEA-1), which is now known to be MASP-3-dependent, and lectin pathwayeffector arm 2 (LEA-2), which is MASP-2-dependent. As used herein,activation of the lectin pathways are assessed using Ca⁺⁺ containingbuffers.

As used herein, the term “classical pathway” refers to complementactivation that is triggered by antibody bound to a foreign particle andrequires binding of the recognition molecule C1q.

As used herein, the term “HTRA-1” refers to the serine peptidaseHigh-temperature requirement serine protease A.

As used herein, the term “MASP-3 inhibitory agent” refers to any agentthat directly inhibits MASP-3-dependent complement activation, includingagents that bind to or directly interact with MASP-3, including MASP-3antibodies and MASP-3 binding fragments thereof, natural and syntheticpeptides, competitive substrates, small-molecules, expression inhibitorsand isolated natural inhibitors, and also encompasses peptides thatcompete with MASP-3 for binding to another recognition molecule (e.g.,MBL, CL-11, H-ficolin, M-ficolin, or L-ficolin) in the lectin pathway.In one embodiment, the MASP-3 inhibitory agent is specific to MASP-3,and does not bind to MASP-1 or MASP-2. An inhibitory agent that directlyinhibits MASP-3 can be referred to as a direct MASP-3 inhibitory agent(e.g., a MASP-3 antibody), while an inhibitory agent that indirectlyinhibits MASP-3 can be referred to as an indirect MASP-3 inhibitoryagent (e.g., a MASP-1 antibody that inhibits MASP-3 activation). Anexample of a direct MASP-3 inhibitory agent is a MASP-3 specificinhibitory agent, such as a MASP-3 inhibitory agent that specificallybinds to a portion of human MASP-3 (

-   SEQ ID NO:2) with a binding affinity of at least 10 times greater    than to other components in the complement system. Another example    of a direct MASP-3 inhibitory agent is a high affinity MASP-3    antibody that specifically binds to the serine protease domain of    human MASP-3 (SEQ ID NO:2), with an affinity of less than 500 pM and    does not bind to human MASP-1 (SEQ ID NO:8). In one embodiment, a    MASP-3 inhibitory agent indirectly inhibits MASP-3 activity, such    as, for example, an inhibitor of MASP-3 activation, including an    inhibitor of MASP-1-mediated MASP-3 activation (e.g., a MASP-1    antibody or MASP-1 binding fragments thereof, natural and synthetic    peptides, small-molecules, expression inhibitors and isolated    natural inhibitors, and also encompasses peptides that compete with    MASP-1 for binding to MASP-3). In a preferred embodiment, a MASP-3    inhibitory agent, such as an antibody or antigen-binding fragment    thereof or antigen binding peptide inhibits MASP-3-mediated    maturation of factor D. In another embodiment, a MASP-3 inhibitory    agent inhibits MASP-3-mediated activation of factor B. MASP-3    inhibitory agents useful in the method of the invention may reduce    MASP-3-dependent complement activation by greater than 10%, such as    greater than 20%, greater than 50%, or greater than 90%. In one    embodiment, the MASP-3 inhibitory agent reduces MASP-3-dependent    complement activation by greater than 90% (i.e., resulting in MASP-3    complement activation of only 10% or less). It is expected that    MASP-3 inhibition will block, in full or in part, both LEA-1-related    lysis and opsonization and lectin-independent conversion of factor B    and factor D-related lysis and opsonization.

In one embodiment, a high affinity MASP-3 inhibitory antibody binds tothe serine protease domain of MASP-3 (amino acid residues 450 to 728 ofSEQ ID NO:2) with an affinity of less than 500 pM (e.g., less than 250pM, less than 100 pM, less than 50 pM, or less than 10 pM) and inhibitthe alternative pathway of complement activation in the blood of amammalian subject by at least 50% (e.g., at least 60%, or at least 70%,or at least 80%, or at least 90%, or at least 95% or greater).

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a polypeptide, through at least one epitoperecognition site located in the variable region (also referred to hereinas the variable domain) of the immunoglobulin molecule.

As used herein, the term “antibody” encompasses antibodies and antibodyfragments thereof, derived from any antibody-producing mammal (e.g.,mouse, rat, rabbit, and primate including human), or from a hybridoma,phage selection, recombinant expression or transgenic animals (or othermethods of producing antibodies or antibody fragments), thatspecifically bind to a target polypeptide, such as, for example, MASP-1,MASP-2 or MASP-3 polypeptides or portions thereof. It is not intendedthat the term “antibody” is limited as regards to the source of theantibody or the manner in which it is made (e.g., by hybridoma, phageselection, recombinant expression, transgenic animal, peptide synthesis,etc). Exemplary antibodies include polyclonal, monoclonal andrecombinant antibodies; pan-specific, multispecific antibodies (e.g.,bispecific antibodies, trispecific antibodies); humanized antibodies;murine antibodies; chimeric, mouse-human, mouse-primate, primate-humanmonoclonal antibodies; and anti-idiotype antibodies, and may be anyintact antibody or fragment thereof. As used herein, the term “antibody”encompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof,such as a single variable region antibody (dAb),or other known antibody fragments such as Fab, Fab′, F(ab′)₂, Fv and thelike, single chain (ScFv), synthetic variants thereof, naturallyoccurring variants, fusion proteins comprising an antibody portion withan antigen-binding fragment of the required specificity, humanizedantibodies, chimeric antibodies, bi-specific antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen-binding site or fragment (epitope recognition site) of therequired specificity.

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an epitope. Monoclonal antibodies are highlyspecific for the target antigen. The term “monoclonal antibody”encompasses not only intact monoclonal antibodies and full-lengthmonoclonal antibodies, but also fragments thereof (such as Fab, Fab′,F(ab′)₂, Fv), single chain (ScFv), variants thereof, fusion proteinscomprising an antigen-binding portion, humanized monoclonal antibodies,chimeric monoclonal antibodies, and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen-binding fragment(epitope recognition site) of the required specificity and the abilityto bind to an epitope. It is not intended to be limited as regards thesource of the antibody or the manner in which it is made (e.g., byhybridoma, phage selection, recombinant expression, transgenic animals,etc.). The term includes whole immunoglobulins as well as the fragmentsetc. described above under the definition of “antibody”.

As used herein, the term “antibody fragment” refers to a portion derivedfrom or related to a full-length antibody, such as, for example, aMASP-1, MASP-2 or MASP-3 antibody, generally including the antigenbinding or variable region thereof Illustrative examples of antibodyfragments include Fab, Fab′, F(ab)₂, F(ab′)₂ and Fv fragments, scFvfragments, diabodies, linear antibodies, single-chain antibody moleculesand multispecific antibodies formed from antibody fragments.

In certain embodiments, antibodies and antigen-binding fragments thereofas described herein include a heavy chain (VH) and a light chain (VL)complementarity-determining region (“CDR”) set, respectively interposedbetween a heavy chain and a light chain framework region (FR) set whichprovide support to the CDRs and define the spatial relationship of theCDRs relative to each other. As used herein, the term “CDR set” refersto the three hypervariable regions of a heavy or light chain V region.Proceeding from the N-terminus of a heavy or light chain, these regionsare denoted as “CDR1,” “CDR2,” and “CDR3” respectively. Anantigen-binidng site, therefore, includes six CDRs, comprising the CDRset from each of a heavy and a light chain V region.

As used herein, the term “I/R set” refers to the four flanking aminoacid sequences which frame the CDRs of a CDR set of a heavy or lightchain V region. Some I/R residues may contact bound antigen; however,I/Rs are primarily responsible for folding the V region into theantigen-binding site, particularly the I/R residues directly adjacent tothe CDRs. Within I/Rs, certain amino acid residues and certainstructural features are very highly conserved. In this regard, all Vregion sequences contain an internal disulfide loop of around 90 aminoacid residues. With the V regions fold into a binding-site, the CDRs aredisplayed as projecting loop motifs which form an antigen-bindingsurface. It is generally recognized that there are conserved structuralregions of I/Rs which influence the folded shape of the CDR loops intocertain “canonical” structures-regardless of the precise CDR amino acidsequence.

The structures and locations of immunoglobulin variable regions may bedetermined by reference to Kabat, E. A., et al., Sequences of Proteinsof Immunological Interest, 4^(th) Edition, US Department of Health andHuman Services, 1987, and updates thereof, now available on the Internet(immuno.bme.nwu.edu.).

As used herein, a “single-chain Fv” or “scFv” antibody fragmentcomprises the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains, which enables the scFv to form the desired structure forantigen binding.

As used herein, a “chimeric antibody” is a recombinant protein thatcontains the variable domains and complementarity-determining regionsderived from a non-human species (e.g., rodent) antibody, while theremainder of the antibody molecule is derived from a human antibody. Insome embodiments, a chimeric antibody is comprised of an antigen-bindingfragment of a MASP-3 inhibitory antibody operably linked or otherwisefused to a heterologous Fc portion of a different antibody. In someembodiments, the heterologous Fc domain may be from a different Ig classfrom the parent antibody, including IgA (including subclasses IgAl andIgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4)and IgM.

As used herein, a “humanized antibody” is a chimeric molecule, generallyprepared using recombinant techniques, having an antigen-binding sitederived from an immunoglobulin from a non-human species and theremaining immunoglobulin structure of the molecule based upon thestructure and/or sequence of a human immunoglobulin. The antigen-bindingsite may comprise either complete variable regions fused onto constantdomains or only the CDRs grafted onto appropriate framework regions inthe variable domains. Epitope binding sites may be wild type or may bemodified by one or more amino acid substitutions. Another approachfocuses not only on providing human-derived constant regions, but alsoon modifying the variable regions as well so as to reshape them asclosely as possible to human form. In some embodiments, humanizedantibodies preserve all CDR sequences (for example, a humanized mouseantibody which contains all six CDRs from the mouse antibodies). Inother embodiments, humanized antibodies have one or more CDRs (one, two,three, four, five, six) which are altered with respect to the originalantibody, which are also termed one or more CDRs “derived from” one ormore CDRs from the original antibody.

An antibody “specifically binds” to a target if it binds with greateraffinity and/or avidity that it binds to other substances. In oneembodiment, the antibody, or antigen-binding fragment thereof,specifically binds to the serine protease domain of human MASP-3 (aminoacid residues 450 to 728 of SEQ ID NO:2). In one embodiment, theantibody, or antigen-binding fragment thereof, specifically binds to oneor more of the epitopes described in TABLE 4, TABLE 28 or shown in FIG.62.

As used herein, the term “mannan-binding lectin” (“MBL”) is equivalentto mannan-binding protein (“MBP”).

As used herein, the “membrane attack complex” (“MAC”) refers to acomplex of the terminal five complement components (C5b combined withC6, C7, C8 and C9) that inserts into and disrupts membranes (alsoreferred to as C5b-9).

As used herein, “a subject” includes all mammals, including withoutlimitation humans, non-human primates, dogs, cats, horses, sheep, goats,cows, rabbits, pigs and rodents.

As used herein, the amino acid residues are abbreviated as follows:alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine(Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q),glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L),lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline(Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine(Tyr;Y), and valine (Val;V).

In the broadest sense, the naturally occurring amino acids can bedivided into groups based upon the chemical characteristic of the sidechain of the respective amino acids. By “hydrophobic” amino acid ismeant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By“hydrophilic” amino acid is meant either Gly, Asn, Gln, Ser, Thr, Asp,Glu, Lys, Arg or His. This grouping of amino acids can be furthersubclassed as follows. By “uncharged hydrophilic” amino acid is meanteither Ser, Thr, Asn or Gln. By “acidic” amino acid is meant either Gluor Asp. By “basic” amino acid is meant either Lys, Arg or His.

As used herein the term “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine.

The term “oligonucleotide” as used herein refers to an oligomer orpolymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) ormimetics thereof. This term also covers those oligonucleobases composedof naturally-occurring nucleotides, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring modifications.

As used herein, an “epitope” refers to the site on a protein (e.g., ahuman MASP-3 protein) that is bound by an antibody. “Overlappingepitopes” include at least one (e.g., two, three, four, five, or six)common amino acid residue(s), including linear and non-linear epitopes.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably and mean any peptide-linked chain of amino acids,regardless of length or post-translational modification. The MASP-3proteins described herein can contain or be wild-type proteins or can bevariants that have not more than 50 (e.g., not more than one, two,three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35,40, or 50) conservative amino acid substitutions. Conservativesubstitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine, and leucine; asparticacid and glutamic acid; asparagine, glutamine, serine and threonine;lysine, histidine and arginine; and phenylalanine and tyrosine.

In some embodiments, the human MASP-3 protein can have an amino acidsequence that is, or is greater than, 70 (e.g., 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100) % identical to the human MASP-3 proteinhaving the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments, peptide fragments can be at least 6 (e.g., at least7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, or 600 or more) amino acid residues in length (e.g., at least6 contiguous amino acid residues in SEQ ID NO:2). In some embodiments,an antigenic peptide fragment of a human MASP-3 protein is fewer than500 (e.g., fewer than 450, 400, 350, 325, 300, 275, 250, 225, 200, 190,180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65,60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6) amino acid residues in length(e.g., fewer than 500 contiguous amino acid residues in SEQ ID NO:2.

In some embodiments, in the context of generating an antibody that bindsMASP-3, the peptide fragments are antigenic and retain at least 10%(e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 50%, at least 55%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least98%, at least 99%, at least 99.5%, or 100% or more) of the ability ofthe full-length protein to induce an antigenic response in a mammal (seebelow under “Methods for Producing an Antibody”).

Percent (%) amino acid sequence identity is defined as the percentage ofamino acids in a candidate sequence that are identical to the aminoacids in a reference sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Alignment for purposes of determining percent sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full-length of thesequences being compared can be determined by known methods.

In representative embodiments, the human MASP-3 protein (SEQ ID NO:2) isencoded by the cDNA sequence set forth as SEQ ID NO:1. Those skilled inthe art will recognize that the cDNA sequence disclosed in SEQ ID NO:1represents a single allele of human MASP-3, and that allelic variationand alternative splicing are expected to occur. Allelic variants of thenucleotide sequences shown in SEQ ID NO:1, including those containingsilent mutations and those in which mutations result in amino acidsequence changes, are within the scope of the present invention. Allelicvariants of the MASP-3 sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures,or may be identified by homology comparison search (e.g., BLASTsearching) of databases containing such information.

As used herein, an “isolated nucleic acid molecule” is a nucleic acidmolecule (e.g., a polynucleotide) that is not integrated in the genomicDNA of an organism. For example, a DNA molecule that encodes a growthfactor that has been separated from the genomic DNA of a cell is anisolated DNA molecule. Another example of an isolated nucleic acidmolecule is a chemically-synthesized nucleic acid molecule that is notintegrated in the genome of an organism. A nucleic acid molecule thathas been isolated from a particular species is smaller than the completeDNA molecule of a chromosome from that species.

As used herein, a “nucleic acid molecule construct” is a nucleic acidmolecule, either single- or double-stranded, that has been modifiedthrough human intervention to contain segments of nucleic acid combinedand juxtaposed in an arrangement not existing in nature.

As used herein, an “expression vector” is a nucleic acid moleculeencoding a gene that is expressed in a host cell. Typically, anexpression vector comprises a transcription promoter, a gene, and atranscription terminator. Gene expression is usually placed under thecontrol of a promoter, and such a gene is said to be “operably linkedto” the promoter. Similarly, a regulatory element and a core promoterare operably linked if the regulatory element modulates the activity ofthe core promoter.

As used herein, the term “about” as used herein is meant to specify thatthe specific value provided may vary to a certain extent, such as avariation in the range of ±10%, preferably ±5%, most preferably ±2% areincluded in the given value. Where ranges are stated, the endpoints

Where ranges are stated, the endpoints are included within the rangeunless otherwise stated or otherwise evident from the context.

As used herein the singular forms “a”, “an” and “the” include pluralaspects unless the context clearly dictates otherwise. Thus, forexample, reference to “an excipient” includes a plurality of suchexcipients and equivalents thereof known to those skilled in the art,reference to “an agent” includes one agent, as well as two or moreagents; reference to “an antibody” includes a plurality of suchantibodies and reference to “a framework region” includes reference toone or more framework regions and equivalents thereof known to thoseskilled in the art, and so forth.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise. It iscontemplated that any embodiment discussed in this specification can beimplemented with respect to any method, kit, reagent, or composition ofthe invention, and vice versa. Furthermore, compositions of theinvention can be used to achieve methods of the invention.

II. THE LECTIN PATHWAY: A NEW UNDERSTANDING

i. Overview: the Lectin Pathway has been Redefined

As described herein, the inventors have made the surprising discoverythat the lectin pathway of complement has two effector arms to activatecomplement, both driven by lectin pathway activation complexes formed ofcarbohydrate recognition components (MBL, CL-11 and ficolins): i) theeffector arm formed by the lectin pathway-associated serine proteasesMASP-1 and MASP-3, referred to herein as “lectin pathway effector arm 1”or “LEA-1”; and (ii) the MASP-2 driven activation effector arm, referredto herein as “lectin pathway effector arm 2”, or “LEA-2”. Both LEA-41and LEA-2 can effect lysis and/or opsonization.

It has also been determined that lectin-independent conversion of factorB by MASP-3 and lectin-independent conversion of factor D by HTRA-1,MASP-1 and MASP-3, which both can occur in the absence of Ca⁺⁺, commonlylead to the conversion of C3bB to C3bBb and of pro-factor D to factor D.Therefore, inhibiting MASP-3 can inhibit both LEA-1 and thelectin-independent activation of factor B and/or factor D, which canresult in the inhibition of lysis and/or opsonization.

FIG. 1 illustrates this new understanding of the pathways of complementactivation. As shown in FIG. 1, LEA-1 is driven by lectin-bound MASP-3,which can activate the zymogen of factor D to its active form and/orcleave the C3b- or C3b(H₂O)-bound factor B, leading to conversion of theC3bB zymogen complex into its enzymatically active form C3bBb. Activatedfactor D, generated by MASP-3, can also convert the C3bB or C3b(H₂O)zymogen complexes into their enzymatically active form. MASP-1 iscapable of rapid self-activation, whereas MASP-3 is not. In many cases,MASP-1 is the activator of MASP-3.

While in many examples lectins (i.e., MBL, CL-11 or ficolins) can directactivity to cellular surfaces, FIG. 1 also outlines thelectin-independent functions of MASP-3, MASP-1, and HTRA-1 in factor Bactivation and/or factor D maturation. As with the lectin-associatedform of MASP-3 in LEA-1, the lectin-independent form of MASP-3 iscapable of mediating conversion of C3bB or C3b(H₂O) to C3bBb (see alsoFIGS. 29 and 30) and pro-factor D to factor D (see FIG. 32). MASP-1 (seealso FIG. 32) and the non-MASP-related protein HTRA-1 can also activatefactor D (Stanton et al., Evidence That the HTRA1 Interactome InfluencesSusceptibility to Age-Related Macular Degeneration, presented at TheAssociation for Research in Vision and Ophthalmology 2011 conference onMay 4, 2011) in a manner in which no lectin component is required.

Thus, MASP-1 (via LEA-1 and lectin-independent forms), MASP-3 (via LEA-1and lectin-independent forms), and HTRA-1 (lectin-independent only) arecapable of either direct or indirect activation at one or more pointsalong a MASP-3-factor D-factor B axis. In doing so, they generate C3bBb,the C3 convertase of the alternative pathway, and they stimulate theproduction and deposition of C3b on microbial surfaces. C3b depositionplays a critical role in opsonization, labeling the surfaces of microbesfor destruction by host phagocytic cells, such as macrophages. As anexample herein (FIGS. 28A and 28B), MASP-3 is critical for opsonizationof S. aureus. C3b deposition occurs rapidly on S. aureus exposed tohuman serum in a MASP-3-dependent fashion (FIGS. 28A and 28B).

The contributions of LEA-1 and the lectin-independent functions ofMASP-3, MASP-1, or HTRA-1 are not limited to opsonization, however. Asdiagrammed in FIG. 1, these three components can also cause cell lysisby indirect or direct activation of factor B, and the production of C3b.These components form complexes that generate the alternative pathway C5convertase, C3bBb(C3b)n. As described further herein, the requirementfor MASP-3 and MBL, but not MASP-2 (and, therefore, not LEA-2 in thisexample), in the lysis of N. meningitidis (see FIGS. 11, 12 and 13)demonstrates a role for LEA-1 in lysis. In summary, the opsonizationresults obtained from the S. aureus studies and the lysis resultsobserved in the N. meningitidis studies support the role of LEA-1 inboth processes (as diagrammed in FIG. 1). Furthermore, these studiesdemonstrate that both opsonization and lysis can result from theconversion of C3bB or C3b(H₂O) and/or of pro-factor D to factor D;therefore, both processes can be outcomes of the lectin-independentroles of MASP-3, MASP-1, or HTRA-1. Thus, the model developed by theinventors in FIG. 1 supports the use of inhibitors of principallyMASP-3, but also MASP-1 and/or HTRA-1, to block opsonization and/orlysis and to treat pathologies caused by dysregulation of theseprocesses.

1. Lectin Pathway Effector Arm (LEA-1)

The first effector arm of the lectin pathway, LEA-1, is formed by thelectin pathway-associated serine proteases MASP-1 and MASP-3. Asdescribed herein, the inventors have now shown that, in the absence ofMASP-3 and in the presence of MASP-1, the alternative pathway is noteffectively activated on surface structures. These results demonstratethat MASP-3 plays a previously undisclosed role in initiating thealternative pathway, and this is confirmed using the MASP-3-deficient3MC serum obtained from patients with the rare 3MC autosomal recessivedisorder (Rooryck C, et al., Nat Genet. 43(3):197-203 (2011)) withmutations that render the serine protease domain of MASP-3dysfunctional. Based on these novel findings, it is expected thatcomplement activation involving the alternative pathway, asconventionally defined, is MASP-3-dependent. In fact, MASP-3, and itsactivation of LEA-1, may represent the hitherto elusive initiator of thealternative pathway.

As further described in Examples 1-4 herein, in MASP-2-deficient sera,the inventors observed a higher activity of lectin-dependent alternativepathway activation resulting in a higher bactericidal activity (i.e.,lytic activity) against N. meningitidis. While not wishing to be boundby any particular theory, it is believed that in absence of MASP-2,MASP-1-bearing carbohydrate recognition complexes are more likely tobind close to MASP-3-bearing carbohydrate recognition complexes toactivate MASP-3. It is known that, in many instances, activation ofMASP-3 is dependent on MASP-1 activity, as MASP-3 is not anauto-activating enzyme and very often requires the activity of MASP-1 tobe converted from its zymogen form into its enzymatically active form.MASP-1 (like MASP-2) is an auto-activating enzyme, while MASP-3 does notauto-activate and, in many instances, needs the enzymatic activity ofMASP-1 to be converted into its enzymatically active form. See, ZundelS, et al., J Immunol., 172(7):4342-50 (2004). In absence of MASP-2, alllectin pathway recognition complexes are either loaded with MASP-1 orMASP-3. Therefore, the absence of MASP-2 facilitates the MASP-1-mediatedconversion of MASP-3 into its enzymatically active form. Once MASP-3 isactivated, activated MASP-3 initiates alternative pathway activation,now referred to as “LEA-1” activation, through a MASP-3-mediatedconversion of C3bB to C3bBb and/or conversion of pro-factor D to factorD. C3bBb, also referred to as the alternative pathway C3 convertase,cleaves additional C3 molecules yielding deposition of opsonic C3bmolecules. If several C3b fragments bind in close proximity to the C3bBbconvertase complex, this results in the formation of the alternativepathway C5 convertase C3bBb(C3b)n, which promotes formation of MAC.Additionally, C3b molecules deposited on the surface form new sites forfactor B binding, which can now be cleaved by factor D and/or MASP-3 toform additional sites where alternative pathway C3 and C5 convertasecomplexes can be formed. This latter process is needed for effectivelysis and does not require lectins once the initial C3b deposition hasoccurred. A recent publication (Iwaki D. et al., J Immunol 187(7):3751-8(2011)) as well as data generated from the inventors (FIG. 30)demonstrate that the alternative pathway C3 convertase zymogen complexC3bB is converted into its enzymatically active form by activatedMASP-3. The inventors now have discovered that the MASP-3-mediatedcleavage of factor B represents a subcomponent of the newly describedLEA-1, which promotes lectin-dependent formation of the alternativepathway C3 convertase C3bBb.

2. Lectin Pathway Effector Arm (LEA-2)

The second effector arm of the lectin pathway, LEA-2, is formed by thelectin pathway-associated serine protease MASP-2. MASP-2 is activatedupon binding of the recognition components to their respective pattern,and may also be activated by MASP-1, and subsequently cleaves thecomplement component C4 into C4a and C4b. After the binding of thecleavage product C4b to plasma C2, C4b-bound C2 becomes substrate of asecond MASP-2-mediated cleavage step which converts C4b-bound C2 intothe enzymatically active complex C4bC2a and a small C2b cleavagefragment. C4b2a is the C3-converting C3 convertase of the lectinpathway, converting the abundant plasma component C3 into C3a and C3b.C3b binds to any surface in close proximity via a thioester bond. Ifseveral C3b fragments bind in close proximity to the C3 convertasecomplex C4b2a, this convertase alters its specificity to convert C5 intoC5b and C5a, forming the C5 convertase complex C4b2a(C3b)n. While thisC5 convertase can initiate formation of MAC, this process is thought tobe insufficiently effective to promote lysis on its own. Rather, theinitial C3b opsonins produced by LEA-2 form the nucleus for theformation of new alternative pathway C3 convertase and C5 convertasesites, which ultimately lead to abundant MAC formation and lysis. Thislatter event is mediated by factor D activation of factor B associatedwith LEA-2-formed C3b, and hence is dependent on LEA-1 by virtue of theessential role for MASP-1 in the maturation of factor D. There is also aMASP-2-dependent C4-bypass activation route to activate C3 in theabsence of C4, which plays an important role in the pathophysiology ofischemia-reperfusion injury, since C4-deficient mice are not protectedfrom ischemia-reperfusion injury while MASP-2-deficient mice are(Schwaeble et al., PNAS, 2011 supra). LEA-2 is also tied to thecoagulation pathway, involving the cleavage of prothrombin to thrombin(common pathway) and also the cleavage of factor XII (Hageman factor) toconvert into its enzymatically active form XIIa. Factor XIIa in turncleaves factor XI to XIa (intrinsic pathway). The intrinsic pathwayactivation of the clotting cascade leads to fibrin formation, which isof critical importance for thrombus formation.

FIG. 1 illustrates the new understanding of the lectin pathway andalternative pathway based on the results provided herein. FIG. 1delineates the role of LEA-2 in both opsonization and lysis. WhileMASP-2 is the initiator of “downstream” C3b deposition (and resultantopsonization) in multiple lectin-dependent settings physiologically(FIGS. 18A, 18B, 18C), it also plays a role in lysis of serum-sensitivebacteria. As illustrated in FIG. 1, the proposed molecular mechanismresponsible for the increased bactericidal activity of MASP-2-deficientor MASP-2-depleted serum/plasma for serum-sensitive pathogens such as N.meningitidis is that, for the lysis of bacteria, lectin pathwayrecognition complexes associated with MASP-1 and MASP-3 have to bind inclose proximity to each other on the bacterial surface, thereby allowingMASP-1 to cleave MASP-3. In contrast to MASP-1 and MASP-2, MASP-3 is notan auto-activating enzyme, but, in many instances, requiresactivation/cleavage by MASP-1 to be converted into its enzymaticallyactive form.

As further shown in FIG. 1, activated MASP-3 can then cleave C3b-boundfactor B on the pathogen surface to initiate the alternative activationcascade by formation of the enzymatically active alternative pathway C3and C5 convertases C3bBb and C3bBb(C3b)n, respectively. MASP-2-bearinglectin-pathway activation complexes have no part in the activation ofMASP-3 and, in the absence of or after depletion of MASP-2, all-lectinpathway activation complexes will either be loaded with MASP-1 orMASP-3. Therefore, in the absence of MASP-2, the likelihood is markedlyincreased that on the microbial surface MASP-1- and MASP-3-bearinglectin-pathway activation complexes will come to sit in close proximityto each other, leading to more MASP-3 being activated and therebyleading to a higher rate of MASP-3-mediated cleavage of C3b-bound factorB to form the alternative pathway C3 and C5 convertases C3bBb andC3bBb(C3b)n on the microbial surface. This leads to the activation ofthe terminal activation cascades C5b-C9 that forms the Membrane AttackComplex, composed of surface-bound C5b associated with C6, C5bC6associated with C7, C5bC6C7 associated with C8, and C5bC6C7C8, leadingto the polymerization of C9 that inserts into the bacterial surfacestructure and forms a pore in the bacterial wall, which will lead toosmolytic killing of the complement-targeted bacterium.

The core of this novel concept is that the data provided herein clearlyshow that the lectin pathway activation complexes drive the followingtwo distinct activation routes, as illustrated in FIG. 1:

i) LEA-1: A MASP-3-dependent activation route that initiates and drivesactivation of complement by generating the alternative pathwayconvertase C3bBb through initial cleavage and activation of factor B onactivator surfaces, which will then catalyze C3b deposition andformation of the alternative pathway convertase C3bBb. The MASP-3-drivenactivation route plays an essential role in the opsonization and lysisof microbes and drives the alternative pathway on the surface ofbacteria, leading to optimal rates of activation to generate membraneattack complexes; and

ii) LEA-2: A MASP-2-dependent activation route leading to the formationof the lectin pathway C3 convertase C4b2a and, upon accumulation of theC3 cleavage product C3b, subsequently to the C5 convertase C4b2a(C3b)n.In the absence of complement C4, MASP-2 can form an alternative C3convertase complex which involves C2 and clotting factor XI.

In addition to its role in lysis, the MASP-2-driven activation routeplays an important role in bacterial opsonization leading to microbesbeing coated with covalently bound C3b and cleavage products thereof(i.e., iC3b and C3dg), which will be targeted for the uptake and killingby C3 receptor-bearing phagocytes, such as granulocytes, macrophages,monocytes, microglia cells and the reticuloendothelial system. This isthe most effective route of clearance of bacteria and microorganismsthat are resistant to complement lysis. These include most of thegram-positive bacteria.

In addition to LEA-1 and LEA-2, there is the potential forlectin-independent activation of factor D by MASP-3, MASP-1 and/orHTRA-1, and there is also the potential for lectin-independentactivation of factor B by MASP-3.

While not wishing to be bound by any particular theory, it is believedthat each of (i) LEA-1, (ii) LEA-2 and (iii) lectin-independentactivation of factor B and/or factor D lead to opsonization and/or theformation of MAC with resultant lysis.

ii. Background of MASP-1, MASP-2 and MASP-3

Three mannan-binding lectin-associated serine proteases (MASP-1, MASP-2and MASP-3) are presently known to be associated in human serum with themannan-binding lectin (MBL). Mannan-binding lectin is also called‘mannose-binding protein’ or ‘mannose-binding lectin’ in the recentliterature. The MBL-MASP complex plays an important role in innateimmunity by virtue of the binding of MBL to carbohydrate structurespresent on a wide variety of microorganisms. The interaction of MBL withspecific arrays of carbohydrate structures brings about the activationof the MASP proenzymes which, in turn, activate complement by cleavingthe complement components C4 and C2 to form the C3 convertase C4b2b(Kawasaki et al., J. Biochem 106:483-489 (1989); Matsushita & Fujita, J.Exp Med. 176:1497-1502 (1992); Ji et al., Immunol 150:571-578 (1993)).

The MBL-MASP proenzyme complex was, until recently, considered tocontain only one type of protease (MASP-1), but it is now clear thatthere are two other distinct proteases (i.e., MASP-2 and MASP-3)associated with MBL (Thiel et al., Nature 386:506-510 (1997); Dahl etal., Immunity 15:127-135 (2001)), as well as an additional serum proteinof 19 kDa, referred to as “MAp19” or “sMAP” (Stover et al., J. Immunol162:3481-3490 (1999); Stover et al., J. Immunol 163:6848-6859 (1999);Takahashi et al., Int. Immunol 11:859-63 (1999)).

MAp19 is an alternatively spliced gene product of the structural genefor MASP-2 and lacks the four C-terminal domains of MASP-2, includingthe serine endopeptidase domain. The abundantly expressed truncated mRNAtranscript encoding MAp19 is generated by an alternativesplicing/polyadenylation event of the MASP-2 gene. By a similarmechanism, the MASP-1/3 gene gives rise to three major gene products,the two serine proteases MASP-1 and MASP-3 and a truncated gene productof 44 kDa referred to as “MAp44” (Degn et al., J. Immunol 183(11):7371-8(2009); Skjoedt et al., J Biol Chem 285:8234-43 (2010)).

MASP-1 was first described as the P-100 protease component of the serumRa-reactive factor, which is now recognized as being a complex composedof MBL plus MASP (Matsushita et al., Collectins and Innate Immunity,(1996); Ji et al., J Immunol 150:571-578 (1993). The ability of anMBL-associated endopeptidase within the MBL-MASPs complex to act on thecomplement components C4 and C2 in a manner apparently identical to thatof the C1s enzyme within the C1q-(C1r)₂-(C1s)₂ complex of the classicalpathway of complement suggests that there is a MBL-MASPs complex whichis functionally analogous to the C1q-(C1r)₂-(C1s)₂ complex. TheC1q-(C1r)₂-(C1s)₂ complex is activated by the interaction of C1q withthe Fc regions of antibody IgG or IgM present in immune complexes. Thisbrings about the autoactivation of the C1r proenzyme which, in turn,activates the C1s proenzyme which then acts on complement components C4and C2.

The stoichiometry of the MBL-MASPs complex differs from the one foundfor the C1q-(C1r)₂-(C1s)₂ complex in that different MBL oligomers appearto associate with different proportions of MASP-1/MAp19 or MASP-2/MASP-3(Dahl et al., Immunity 15:127-135 (2001). The majority of MASPs andMAp19 found in serum are not complexed with MBL (Thiel et al., J Immunol165:878-887 (2000)) and may associate in part with ficolins, a recentlydescribed group of lectins having a fibrinogen-like domain able to bindto N-acetylglucosamine residues on microbial surfaces (Le et al., FEBSLett 425:367 (1998); Sugimoto et al., J. Biol Chem 273:20721 (1998)).Among these, human L-ficolin, H-ficolin and M-ficolin associate withMASPs as well as with MAp19 and may activate the lectin pathway uponbinding to the specific carbohydrate structures recognized by ficolins(Matsushita et al., J Immunol 164:2281-2284 (2000); Matsushita et al., JImmunol 168:3502-3506 (2002)). In addition to the ficolins and MBL, anMBL-like lectin collectin, called CL-11, has been identified as a lectinpathway recognition molecule (Hansen et al. J Immunol 185:6096-6104(2010); Schwaeble et al. PNAS 108:7523-7528 (2011)). There isoverwhelming evidence underlining the physiological importance of thesealternative carbohydrate recognition molecules and it is thereforeimportant to understand that MBL is not the only recognition componentof the lectin activation pathway and that MBL deficiency is not to bemistaken for lectin-pathway deficiency. The existence of possibly anarray of alternative carbohydrate-recognition complexes structurallyrelated to MBL may broaden the spectrum of microbial structures thatinitiate a direct response of the innate immune system via activation ofcomplement.

All lectin pathway recognition molecules are characterized by a specificMASPs-binding motif within their collagen-homologous stalk region(Wallis et al. J. Biol Chem 279:14065-14073 (2004)). The MAST-bindingsite in MBLs, CL-11 and ficolins is characterized by a distinct motifwithin this domain: Hyp-Gly-Lys-Xaa-Gly-Pro, where Hyp is hydroxyprolineand Xaa is generally an aliphatic residue. Point mutations in thissequence disrupt MASP binding.

1. Respective Structures, Sequences, Chromosomal Localization and SpliceVariants of MASP-1 and MASP-3

FIG. 2 is a schematic diagram illustrating the domain structure of thehuman MASP-1 polypeptide (SEQ ID NO:8), human MASP-3 polypeptide (SEQ IDNO:2) and human MAp44 polypeptide and the exons encoding the same. Asshown in FIG. 2, the serine proteases MASP-1 and MASP-3 consist of sixdistinct domains arranged as found in C1r and C1s; i.e., (I) anN-terminal C1r/C1s/sea urchin VEGF/bone morphogenic protein (or CUBI)domain; (II) an epidermal growth factor (EGF)-like domain; (III) asecond CUB domain (CUBII); (IV and V) two complement control protein(CCP1 and CCP2) domains; and (VI) a serine protease (SP) domain.

The cDNA-derived amino acid sequences of human and mouse MASP-1 (Sato etal., Int Immunol 6:665-669 (1994); Takada et al., Biochem Biophys ResCommun 196:1003-1009 (1993); Takayama et al., J. Immunol 152:2308-2316(1994)), human, mouse, and rat MASP-2 (Thiel et al., Nature 386:506-510(1997); Endo et al., J Immunol 161:4924-30 (1998); Stover et al., J.Immunol 162:3481-3490 (1999); Stover et al., J. Immunol 163:6848-6859(1999)), as well as human MASP-3 (Dahl et al., Immunity 15:127-135(2001)) indicate that these proteases are serine peptidases having thecharacteristic triad of His, Asp and Ser residues within their putativecatalytic domains (Genbank Accession numbers: human MASP-1: BAA04477.1(SEQ ID NO:8); mouse MASP-1: BAA03944; rat MASP-1: AJ457084; HumanMASP-3:AAK84071 (SEQ ID NO:2); mouse MASP-3: AB049755, as accessed onGenbank on 2/15/2012 (SEQ ID NO:3); rat MASP-3 (SEQ ID NO:4); chickenMASP-3 (SEQ ID NO:5); rabbit MASP-3 (SEQ ID NO:6); and Cynomolgus monkey(SEQ ID NO:7).

As further shown in FIG. 2, upon conversion of the zymogen to the activeform, the heavy chain (alpha, or A chain) and light chain (beta, or Bchain) are split to yield a disulphide-linked A-chain and a smallerB-chain representing the serine protease domain. The single-chainproenzyme MASP-1 is activated (like proenzyme C1r and C1s) by cleavageof an Arg-Ile bond located between the second CCP domain (domain V) andthe serine protease domain (domain VI). Proenzymes MASP-2 and MASP-3 areconsidered to be activated in a similar fashion to that of MASP-1. EachMASP protein forms homodimers and is individually associated with MBLand the ficolins in a Ca'-dependent manner.

The human MASP-1 polypeptide (SEQ ID NO:8) and MASP-3 polypeptide (SEQID NO:2) arise from one structural gene (Dahl et al., Immunity15:127-135 (2001), which has been mapped to the 3q27-28 region of thelong arm of chromosome 3 (Takada et al., Genomics 25:757-759 (1995)).The MASP-3 and MASP-1 mRNA transcripts are generated from the primarytranscript by an alternative splicing/polyadenylation process. TheMASP-3 translation product is composed of an alpha chain, which iscommon to both MASP-1 and MASP-3, and a beta chain (the serine proteasedomain), which is unique to MASP-3. As shown in FIG. 2, the human MASP-1gene encompasses 18 exons. The human MASP-1 cDNA is encoded by exons 2,3, 4, 5, 6, 7, 8, 10, 11, 13, 14, 15, 16, 17 and 18. As further shown inFIG. 2, the human MASP 3 gene encompasses twelve exons. The human MASP-3cDNA (set forth as SEQ ID NO:1) is encoded by exons 2, 3, 4, 5, 6, 7, 8,10, 11 and 12. An alternative splice results in a protein termedMBL-associated protein 44 (“MAp44),” arising from exons 2, 3, 4, 5, 6,7, 8 and 9.

The human MASP-1 polypeptide (SEQ ID NO: 8 from Genbank BAA04477.1) has699 amino acid residues, which includes a leader peptide of 19 residues.When the leader peptide is omitted, the calculated molecular mass ofMASP-1 is 76,976 Da. As shown in FIG. 2, the MASP-1 amino acid sequencecontains four N-linked glycosylation sites. The domains of the humanMASP-1 protein (with reference to SEQ ID NO:8) are shown in FIG. 2 andinclude an N-terminal C1r/C1s/sea urchin VEFG/bone morphogenic protein(CUBI) domain (aa 25-137 of SEQ ID NO:8), an epidermal growthfactor-like domain (aa 139-181 of SEQ ID NO:8), a second CUB domain(CUBIT) (aa 185-296 of SEQ ID NO:8), as well as a tandem of complementcontrol protein (CCP1 aa 301-363 and CCP2 aa 367-432 of SEQ ID NO:8)domains and a serine protease domain (aa 449-694 of SEQ ID NO:8).

The human MASP-3 polypeptide (SEQ ID NO:2, from Genbank AAK84071) has728 amino acid residues (as shown in FIG. 3), which includes a leaderpeptide of 19 residues (shown as the underlined amino acid residues inFIG. 3).

When the leader peptides are omitted, the calculated molecular mass ofMASP-3 is 81,873 Da. As shown in FIG. 2, there are seven N -linkedglycosylation sites in MASP-3. The domains of the human MASP-3 protein(with reference to SEQ ID NO:2) are shown in FIG. 2 and include anN-terminal C1r/C1s/sea urchin VEGF/bone morphogenic protein (CUBI)domain (aa 25-137 of SEQ ID NO:2), an epidermal growth factor-likedomain (aa 139-181 of SEQ ID NO:2), a second CUB domain (CUBII) (aa185-296 of SEQ ID NO:2), as well as a tandem of complement controlprotein (CCP1 aa 299-363 and CCP2 aa 367-432 of SEQ ID NO:2) domains anda serine protease domain (aa 450-728 of SEQ ID NO:2).

The MASP-3 translation product is composed of an alpha chain (heavychain), containing the CUB-1-EGF-CUB-2-CCP-1-CCP-2 domains (alpha chain:aa 1-448 of SEQ ID NO:2) which is common to both MASP-1 and MASP-3, anda light chain (beta chain: aa 449-728 of SEQ ID NO:2), containing theserine protease domain, which is unique to MASP-3.

2. Comparison of MASP-3 Amino Acid Sequences from Various Species

FIG. 4 provides a multi-species alignment of MASP-3 showing a comparisonof full-length MASP-3 protein from human (SEQ ID NO:2), cynomolgusmonkey (SEQ ID NO:7), rat (SEQ ID NO:4), murine (SEQ ID NO:3), chicken(SEQ ID NO:5) and rabbit (SEQ ID NO:6). FIG. 5 provides a multi-speciesalignment of the serine protease (SP) domain from human (aa 450-728 ofSEQ ID NO:2); rabbit (aa 450-728 of SEQ ID NO:6); murine (aa aa455-733of SEQ ID NO:3); rat (aa 455-733 of SEQ ID NO:4) and chicken (aaaa448-730 of SEQ ID NO:5).

As shown in FIG. 4, there is a high level of amino acid sequenceconservation of MASP-3 polypeptide amongst different species,particularly in the SP domain (FIG. 5). As further shown in FIG. 5, thecatalytic triad (H at residue 497; D at residue 553 and S at residue 664with reference to full length human MASP-3 (SEQ ID NO:2) is conservedacross species. TABLE 1 summarizes the percent identity of the MASP-3 SPdomain across species.

TABLE 1 Percent Identity of the MASP-3 SP domain Across Species CynoRabbit Rat Mouse chicken Human 95% 94% 92% 91% 79% Cyno 94% 90% 90% 79%Rabbit 92% 92% 81% Rat 97% 78% mouse 78%

MASP-3 has no proteolytic activity towards C4, C2 or C3 substrates.Conversely, MASP-3 was initially reported to act as an inhibitor of thelectin pathway (Dahl et al., Immunity 15:127-135 (2001)). Thisconclusion may have come about because in contrast to MASP-1 and MASP-2,MASP-3 is not an autoactivating enzyme (Zundel S. et al., J Immunol172:4342-4350 (2004); Megyeri et al., J. Biol. Chem. 288:8922-8934(2013).

Recently, evidence for possible physiological functions of MASP-1 andMASP-3 emerged from transgenic mouse studies using a mouse strain with acombined MASP-1 and MASP-3 deficiency. While MASP-1/3-knockout mice havea functional lectin pathway (Schwaeble et al., PNAS 108:7523-7528(2011)), they appear to lack alternative pathway activity (Takahashi etal., JEM 207(1):29-37 (2010)). Lack of alternative pathway activityappears to be due to a processing defect of complement factor D, whichis necessary for alternative pathway activity. In MASP-1/3 knockoutmice, all factor D is circulating as a proteolytically inactivepro-form, whereas in the serum of normal mice, substantially all offactor D is in the active form. Biochemical analysis suggested thatMASP-1 may be able to convert complement factor D from its zymogen forminto its enzymatically active form (FIG. 32; Takahashi et al., JEM207(1):29-37 (2010)). MASP-3 also cleaves pro-factor D zymogen andproduce active factor D in vitro (FIG. 32; Takahashi et al., JEM207(1):29-37 (2010)). Factor D is present as an active enzyme incirculation in normal individuals, and MASP-1 and MASP-3, as well asHTRA-1, may be responsible for this activation. Furthermore, mice withcombined MBL and ficolin deficiencies still produce normal levels offactor D and have a fully functional alternative pathway. Thus, thesephysiological functions of MASP-1 and MASP-3 do not necessarily involvelectins, and are thus unrelated to the lectin pathway. Recombinant mouseand human MASP-3 also appear to cleave factor B and support C3deposition on S. aureus in vitro (FIG. 29; Iwaki D. et al., J Immunol187(7):3751-8 (2011)).

An unexpected physiological role for MASP-3 has emerged from recentstudies of patients with 3MC syndrome (previously designated theCarnevale, Mingarelli, Malpuech, and Michels syndrome; OMIM#257920).These patients display severe developmental abnormalities, includingcleft palate, cleft lip, cranial malformations and mental retardation.Genetic analysis identified 3MC patients that were homozygous for adysfunctional MASP-3 gene (Rooryck et al., Nat Genet. 43(3):197-203(2011)). Another group of 3MC patients was found to be homozygous for amutation in the MASP-1 gene that leads to the absence of functionalMASP-1 and MASP-3 proteins. Yet another group of 3MC patients lacked afunctional CL-11 gene. (Rooryck et al., Nat Genet. 43(3):197-203(2011)). Thus, the CL-11 MASP-3 axis appears to play a role duringembryonic development. The molecular mechanisms of this developmentalpathway are unclear. It is unlikely, however, to be mediated by aconventional complement-driven process since individuals withdeficiencies of common complement components C3 do not develop thissyndrome. Thus, prior to the discovery of the instant inventors, asdescribed herein, a functional role for MASP-3 in lectin-dependentcomplement activation was previously not established.

The structures of the catalytic fragment of MASP-1 and MASP-2 have beendetermined by X-ray crystallography. Structural comparison of MASP-1protease domain with those of other complement proteases revealed thebasis of its relaxed substrate specificity (Dobó et al., J. Immunol183:1207-1214 (2009)). While the accessibility of the substrate bindinggroove of MASP-2 is restricted by surface loops (Harmat et al., J MolBiol 342:1533-1546 (2004)), MASP-1 has an open substrate binding pocketwhich resembles that of trypsin rather than other complement proteases.A thrombin-like property of the MASP-1 structure is the unusually large60 amino acid loop (loop B) which may interact with substrates. Anotherinteresting feature of the MASP-1 structure is the internal salt bridgebetween the S1 Asp189 and Arg224. A similar salt bridge can be found inthe substrate binding pocket of factor D, which can regulate itsprotease activity. C1s and MASP-2 have almost identical substratespecificities. Surprisingly, some of the eight surface loops of MASP-2,which determine the substrate specificities, have quite differentconformations compared to those of C 1 s. This means that the twofunctionally related enzymes interact with the same substrates in adifferent manner. The structure of zymogen MASP-2 shows an inactiveprotease domain with disrupted oxyanion hole and substrate bindingpocket (Gál et al., J Biol Chem 280:33435-33444 (2005)). Surprisingly,zymogen MASP-2 shows considerable activity on a large protein substrate,C4. It is likely that the structure of zymogen MASP-2 is quite flexible,enabling the transition between the inactive and the active forms. Thisflexibility, which is reflected in the structure, may play a role in theautoactivation process.

Northern blot analysis indicates that liver is the major source ofMASP-1 and MASP-2 mRNA. Using a 5′ specific cDNA probe for MASP-1, majorMASP-1 transcript was seen at 4.8 kb and a minor one at approximately3.4 kb, both present in human and mouse liver (Stover et al., GenesImmunity 4:374-84 (2003)). MASP-2 mRNA (2.6 kb) and MAp19 mRNA (1.0 kb)are abundantly expressed in liver tissue. MASP-3 is expressed in theliver, and also in many other tissues, including neuronal tissue (LynchN. J. et al., J Immunol 174:4998-5006 (2005)).

A patient with a history of infections and chronic inflammatory diseasewas found to have a mutated form of MASP-2 that fails to form an activeMBL-MASP complex (Stengaard-Pedersen et al., N Engl J Med 349:554-560(2003)). Some investigators have determined that deficiency of MBL leadsto a tendency to frequent infections in childhood (Super et al., Lancet2:1236-1239 (1989); Garred et al., Lancet 346:941-943 (1995) and adecreased resistance to HIV infection (Nielsen et al., Clin Exp Immunol100:219-222 (1995); Garred et al., Mol Immunol 33 (suppl 1):8 (1996)).However, other studies have not demonstrated a significant correlationof low MBL levels with increased infections (Egli et al., PLoS One.8(1):e51983 (2013); Ruskamp et al., J Infect Dis. 198(11):1707-13(2008); Israëls et al., Arch Dis Child Fetal Neonatal Ed. 95(6):F452-61(2010)). While the literature is mixed, deficiency, or non-utilization,of MASP may have an adverse effect on an individual's ability to mountimmediate, non-antibody-dependent defense against certain pathogens.

Supporting Data for the New Understanding, Underscoring TraditionalAssay Conditions that are Devoid of Ca⁺⁺ and Rresults Obtained using aMore Physiological Set of Conditions that Include Ca⁺⁺.

Several independent lines of strong experimental evidence are providedherein pointing to the conclusion that the lectin pathway activationroute of complement activates complement via two independent effectormechanisms: i) LEA-2: a MASP-2-driven path that mediatescomplement-driven opsonisation, chemotaxis (Schwaeble et al., PNAS108:7523-7528 (2011)), and cell lysis, and ii) LEA-1: a novelMASP-3-dependent activation route that initiates complement activationby generating the alternative pathway convertase C3bBb through cleavageand activation of factor B on activator surfaces, which will thencatalyze C3b deposition and formation of the alternative pathwayconvertase C3bBb, which can result in cell lysis as well as microbialopsonization. In addition, as described herein, separatelectin-independent activation of factor B and/or factor D by MASP-1,MASP-3, or HTRA-1, or a combination of any the three, can also lead tocomplement activation via the alternative pathway.

A lectin pathway-dependent MASP-3-driven activation of the alternativepathway appears to contribute to the well-established factor D-mediatedcleavage of C3b-bound factor B to achieve optimal activation rates forcomplement-dependent lysis through the terminal activation cascade tolyse bacterial cells through the formation of C5b-9 membrane attackcomplexes (MAC) on the cellular surface (FIGS. 12-13). This rate-limitedevent appears to require optimal coordination as it is defective in theabsence of MASP-3 functional activity as well as in the absence offactor D functional activity. As described in Examples 1-4 herein, theinventors discovered this MASP-3-dependent lectin pathway function whenstudying the phenotype of MASP-2 deficiency and MASP-2 inhibition inexperimental mouse models of N. menigitidis infection. Gene-targeted,MASP-2-deficient mice and wild-type mice treated with antibody-basedMASP-2 inhibitors were highly resistant to experimental N. meningitidisinfection (see FIGS. 6-10). When the infectious dose was adjusted togive approximately 60% mortality in the wild-type littermates, all ofthe MASP-2-deficient or MASP-2-depleted mice cleared the infection andsurvived (see FIG. 6 and FIG. 10). This extremely high degree ofresistance was reflected in a significant increase of serum bactericidalactivity in MASP-2-deficient or MASP-2-depleted mouse serum. Furtherexperiments showed that this bactericidal activity was dependent onalternative pathway-driven bacterial lysis. Mouse sera deficient offactor B, or factor D, or C3 showed no bactericidal activity towards N.meningitidis indicating that the alternative pathway is essential fordriving the terminal activation cascade. A surprising result was thatmouse sera deficient of MBL-A and MBL-C (both being the lectin-pathwayrecognition molecules that recognize N. meningitidis) as well as mousesera deficient of the lectin pathway-associated serine proteases MASP-1and MASP-3 had lost all bacteriolytic activity towards N. meningitidis(FIG. 13). A recent paper (Takahashi M. et al., JEM 207: 29-37 (2010))and work presented herein (FIG. 32) demonstrate that MASP-1 can convertthe zymogen form of factor D into its enzymatically active form and mayin part explain the loss of lytic activity through the absence ofenzymatically active factor D in these sera. This does not explain thelack of bactericidal activity in MBL-deficient mice since these micehave normal enzymatically active factor D (Banda et al., Mol Immunol49(1-2):281-9 (2011)). Remarkably, when testing human sera from patientswith the rare 3MC autosomal recessive disorder (Rooryck C, et al., NatGenet. 43(3):197-203) with mutations that render the serine proteasedomain of MASP-3 dysfunctional, no bactericidal activity against N.meningitidis was detectable (n.b.: these sera have MASP-1 and factor D,but no MASP-3).

The hypothesis that human serum requires lectin pathway-mediatedMASP-3-dependent activity to develop bactericidal activity is furthersupported by the observation that MBL-deficient human sera also fail tolyse N. meningitidis (FIGS. 11-12). MBL is the only human lectin-pathwayrecognition molecule that binds to this pathogen. Since MASP-3 does notauto-activate, the inventors hypothesize that the higher bacteriolyticactivity in MASP-2-deficient sera could be explained by a favoredactivation of MASP-3 through MASP-1 since, in the absence of MASP-2, alllectin-pathway activation complexes that bind to the bacterial surfacewill be loaded with either MASP-1 or MASP-3. Since activated MASP-3cleaves both factor D (FIG. 32) and factor B to generate theirrespective enzymatically active forms in vitro (FIG. 30 and Iwaki D., etal., J. Immunol.187(7):3751-3758 (2011)), the most likely function ofMASP-3 is to facilitate the formation of the alternative pathway C3convertase (i.e., C3bBb).

While the data for the lectin-dependent role are compelling, multipleexperiments suggest that MASP-3 and MASP-1 are not necessarily obligatedto function in a complex with lectin molecules. Experiments such as thatshown in FIG. 28B demonstrate the ability of MASP-3 to activate thealternative pathway (as demonstrated by C3b deposition on S. aureus)under conditions (i.e., the presence of EGTA) in which complexes withlectin would not be present. FIG. 28A demonstrates that deposition underthese conditions is dependent upon factor B, factor D, and factor P, allcritical components of the alternative pathway. Addtionally, factor Dactivation by MASP-3 and MASP-1 (FIG. 32), and factor B activation byMASP-3 (FIG. 30) can occur in vitro in the absence of lectin. Finally,hemolysis studies of mouse erythrocytes in the presence of human serumdemonstrate a clear role for both MBL and MASP-3 for cell lysis.However, the deficiency of MBL does not completely reproduce theseverity of the deficiency of MASP-3, in contrast to what would beexpected if all functional MASP-3 were complexed with MBL. Thus, theinventors do not wish to be constrained by the notion that all of theroles for MASP-3 (and MASP-1) demonstrated herein can be attributedsolely to function associated with lectin.

The identification of the two effector arms of the lectin pathway, aswell as the possible lectin-independent functions of MASP-1, MASP-3, andHTRA-1, represent novel opportunities for therapeutic interventions toeffectively treat defined human pathologies caused by excessivecomplement activation in the presence of microbial pathogens or alteredhost cells or metabolic deposits. As described herein, the inventorshave now discovered that in the absence of MASP-3 and in the presence ofMASP-1, the alternative pathway is not activated on surface structures(see FIGS. 15-16, 28B, 34-35A,B, 38-39). Since the alternative pathwayis important in driving the rate-limiting events leading to bacteriallysis as well as cell lysis (Mathieson PW, et al., J Exp Vied177(6):1827-3 (1993)), our results demonstrate that activated MASP-3plays an important role in the lytic activity of complement. As shown inFIGS. 12-13, 19-21, 36-37, and 39-40, in serum of 3MC patients lackingMASP-3 but not MASP-1, the lytic terminal activation cascade ofcomplement is defective. The data shown in FIGS. 12 and 13 demonstrate aloss of bacteriolytic activity in absence of MASP-3 and/or MASP-1/MASP-3functional activity. Likewise, the loss of hemolytic activity inMASP-3-deficient human serum (FIGS. 19-21, 36-37 and 39-40), coupledwith the ability to reconstitute hemolysis by adding recombinant MASP-3(FIGS. 39-40), strongly supports the conclusion that activation of thealternative pathway on target surfaces (which is essential to drivecomplement-mediated lysis) depends on the presence of activated MASP-3.Based on the new understanding of the lectin pathway detailed above,alternative pathway activation of target surfaces is thus dependent uponLEA-1, and/or lectin-independent activation of factor B and/or factor D,which is also mediated by MASP-3, and therefore, agents that blockMASP-3-dependent complement activation will prevent alternative pathwayactivation on target surfaces.

The disclosure of the essential role of MASP-3-dependent initiation ofalternative pathway activation implies that the alternative pathway isnot an independent, stand-alone pathway of complement activation asdescribed in essentially all current medical textbooks and recent reviewarticles on complement. The current and widely held scientific view isthat the alternative pathway is activated on the surface of certainparticulate targets (microbes, zymosan, and rabbit erythrocytes) throughthe amplification of spontaneous “tick-over” C3 activation. However, theabsence of any alternative pathway activation in sera of MASP-1 andMASP-3 double-deficient mice and human 3MC patient serum on bothzymosan-coated plates and two different bacteria (N. meningitidis and S.aureus), and the reduction of hemolysis of erythrocytes inMASP-3-deficient sera from human and mouse indicate that initiation ofalternative pathway activation on these surfaces requires functionalMASP-3. The required role for MASP-3 may be either lectin-dependent or-independent, and leads to formation of the alternative pathway C3convertase and C5 convertase complexes, i.e. C3bBb and C3bBb(C3b)n,respectively. Thus, the inventors here disclose the existence of apreviously elusive initiation routes for the alternative pathway. Thisinitiation route is dependent upon (i) LEA-1, a newly discoveredactivation arm of the lectin pathway, and/or (ii) lectin-independentroles of the proteins MASP-3, MASP-1, and HTRA-1.

3. The use of MASP-3 Inhibitory Agents for the Treatment of AlternativePathway-Related Diseases and Conditions.

As described herein, high affinity MASP-3 inhibitory antibodies (e.g.,with a binding affinity of less than 500 pM) which have been shown tocompletely inhibit the alternative pathway in mammalian subjects such asrodents and non-primates at molar concentrations less than theconcentration of the MASP-3 target (e.g., at a molar ratio of from about1:1 to about 2.5:1 (MASP-3 target to mAb) (see in Examples 11-21). Asdescribed in Example 11, a single dose administration of a high affinityMASP-3 inhibitory antibody, mAb 13B1, to mice led to near-completeablation of systemic alternative pathway complement activity for atleast 14 days. As further described in Example 12, in a study conductedin a well-established animal model associated with PNH it wasdemonstrated that mAb 13B1 significantly improved the survival ofPNH-like red blood cells and protected PNH-like red blood cellssignificantly better than did C5 inhibition. As described in Example 13,it was further demonstrated that mAb 13B1 reduced the incidence andseverity of disease in a mouse model of arthritis. The results in thisexample demonstrate that representative high affinity MASP-3 inhibitorymAbs 13B1, 10D12 and 4D5 are highly effective at blocking thealternative pathway in primates. Single dose administration of mAb 13B1,10D12 or 4D5 to cynomolgus monkeys resulted in sustained ablation ofsystemic alternative pathway activity lasting for approximately 16 days.The extent of alternative pathway ablation in cynomolgus monkeys treatedwith high affinity MASP-3 inhibitory antibodies was comparable to thatachieved by factor D blockade in vitro and in vivo, indicating completeblockade of factor D conversion by the MASP-3 inhibitory antibodies.Therefore, high affinity MASP-3 inhibitory mAbs have therapeutic utilityin the treatment of patients suffering from diseases related toalternative pathway hyperactivity

Accordingly, in one aspect the invention provides methods of inhibitingthe alternative pathway in a mammalian subject in need thereofcomprising administering to the subject a composition comprising anisolated monoclonal antibody or antigen-binding fragment thereof thatspecifically binds to the serine protease domain of human MASP-3 (aminoacid residues 450 to 728 of SEQ ID NO:2) with high affinity (having aK_(D) of less than 500 pM), in an amount effective to inhibitsalternative pathway complement activation in the subject. In someembodiments, the subject is suffering from an alternativepathway-related disease or disorder, (i.e., a disease or disorderrelated to alternative pathway hyperactivity), such as for example,paroxysmal nocturnal hemoglobinuria (PNH), age-related maculardegeneration (AMD, including wet and dry AMD), ischemia-reperfusioninjury, arthritis, disseminated intravascular coagulation, thromboticmicroangiopathy (including hemolytic uremic syndrome (HUS), atypicalhemolytic uremic syndrome (aHUS),thrombotic thrombocytopenic purpura(TTP) or transplant-associated TMA), asthma, dense deposit disease,pauci-immune necrotizing crescentic glomerulonephritis, traumatic braininjury, aspiration pneumonia, endophthalmitis, neuromyelitis optica,Behcet's disease, multiple sclerosis, Guillain Barre Syndrome,Alzheimer's disease, Amylotrophic lateral sclerosis (ALS), lupusnephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy,Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy andMyasthenia Gravis, as further described below.

A. THE ROLE OF MASP-3 IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA ANDTHERAPEUTIC METHODS USING MASP-3 INHIBITORY ANTIBODIES, OPTIONALLY INCOMBINATION with MASP-2 INHIBITORY AGENTS

Overview of PNH

Paroxysmal nocturnal hemoglobinuria (PNH), sometimes also referred to asMarchiafava-Micheli syndrome, is an acquired, potentiallylife-threatening disease of the blood. PNH may develop on its own,referred to as “primary PNH” or in the context of other bone marrowdisorders such as aplastic anemia, referred to as “secondary PNH.” Themajority of cases are primary PNH. PNH is characterized bycomplement-induced destruction of red blood cells (hemolysis), low redblood cell counts (anemia), thrombosis and bone marrow failure.Laboratory findings in PNH show changes consistent with intravascularhemolytic anemia: low hemoglobin, raised lactate dehydrogenase, raisedreticulocyte counts (immature red cells released by the bone marrow toreplace the destroyed cells), raised bilirubin (a breakdown product ofhemoglobin), in the absence of autoreactive RBC-binding antibodies as apossible cause.

The hallmark of PNH is the chronic complement-mediated hemolysis causedby the unregulated activation of terminal complement components,including the membrane attack complex, on the surface of circulatingRBCs. PNH RBCs are subject to uncontrolled complement activation andhemolysis due to the absence of the complement regulators CD55 and CD59on their surface (Lindorfer, M. A., et al., Blood 115(11):2283-91(2010), Risitano, et al., Mini-Reviews in Medicinal Chemistry,11:528-535 (2011)). CD55 and CD59 are abundantly expressed on normalRBCs and control complement activation. CD55 acts as a negativeregulator of the alternative pathway, inhibiting the assembly of thealternative pathway C3 convertase (C3bBb) complex and accelerating thedecay of preformed convertase, thus blocking the formation of themembrane attack complex (MAC). CD59 inhibits the complement membraneattack complex directly by binding the C5b678 complex and preventing C9from binding and polymerizing.

While hemolysis and anemia are the dominant clinical features of PNH,the disease is a complex hematologic disorder that further includesthrombosis and bone marrow failure as part of the clinical findings(Risitano et al, Mini Reviews in Med Chem, 11:528-535 (2011)). At themolecular level, PNH is caused by the abnormal clonal expansion ofhematopoietic stem cells lacking a functional PIG A gene. PIG A is anX-linked gene encoding a glycosyl-phosphatidyl inositol transferaserequired for the stable surface expression of GPI-anchored class Aglycoproteins, including CD55 and CD59. For reasons that are presentlyunder investigation, hematopoietic stem cells with a dysfunctional PIG Agene that arise as the result of spontaneous somatic mutations canundergo clonal expansion to the point where their progeny constitute asignificant portion of the peripheral hematopoietic cell pool. Whileboth erythrocyte and lymphocyte progeny of the mutant stem cell clonelack CD55 and CD59, only the RBCs undergo overt lysis after they enterthe circulation.

Current treatment for PNH includes blood transfusion for anemia,anticoagulation for thrombosis and the use of the monoclonal antibodyeculizumab (Soliris®), which protects blood cells against immunedestruction by inhibiting the complement system (Hillmen P. et al., N.Engl. J. Med. 350(6):552-559 (2004)). Eculizumab (Soliris®) is ahumanized monoclonal antibody that targets the complement component C5,blocking its cleavage by C5 convertases, thereby preventing theproduction of C5a and the assembly of MAC. Treatment of PNH patientswith eculizumab has resulted in a reduction of intravascular hemolysis,as measured by lactate dehydrogenase (LDH), leading to hemoglobinstabilization and transfusion independence in about half of the patients(Risitano et al, Mini-Reviews in Medicinal Chemistry, 11(6) (2011)).While nearly all patients undergoing therapy with eculizumab achievenormal or almost normal LDH levels (due to control of intravascularhemolysis), only about one third of the patients reach a hemoglobinvalue about l lgr/dL, and the remaining patients on eculizumab continueto exhibit moderate to severe (i.e., transfusion-dependent) anemia, inabout equal proportions (Risitano A. M. et al., Blood 113:4094-100(2009)). As described in Risitano et al., Mini-Reviews in MedicinalChemistry 11:528-535 (2011), it was demonstrated that PNH patients oneculizumab contained large amounts of C3 fragments bound to their PNHerythrocytes (while untreated patients did not). This finding lead tothe recognition that in Soliris treated PNH patients, PNH RBCs that areno longer hemolyzed due to C5 blockade now can accumulate copiousamounts of membrane-bound C3 fragments, which operate as opsonins,resulting in their entrapment in the reticuloendothelial cells throughspecific C3 receptors and subsequent extravascular hemolysis. Thus,while preventing intravascular hemolysis and the resulting sequelae,eculizumab therapy simply diverts the disposition of these RBCs fromintravascular to extravascular hemolysis, resulting in substantialresidual untreated anemia in many patients (Risitano A. M. et al., Blood113:4094-100 (2009)). Therefore, therapeutic strategies in addition tothe use of eculizumab are needed for those patients developingC3-fragment-mediated extravascular hemolysis, because they continue torequire red cell transfusions. Such C3 fragment targeting approacheshave demonstrated utility in experimental systems (Lindorfer et al.,Blood 115:2283-91, 2010).

Complement-Initiating Mechanisms in PNH

The causal link between defective surface expression of the negativecomplement regulators CD55 and CD59 in PNH, combined with theeffectiveness of eculizumab in preventing intravascular hemolysis,clearly define PNH as a condition mediated by the complement system.While this paradigm is widely accepted, the nature of the eventsinitiating complement activation, and the complement activationpathway(s) involved remain unresolved. Because CD55 and CD59 negativelyregulate the terminal amplification steps in the complement cascadecommon to all complement initiation pathways, deficiency of thesemolecules will lead to exaggerated formation and membrane integration ofmembrane attack complexes, regardless of whether complement activationis initiated by the lectin pathway, by the classical pathway or byspontaneous turnover of the alternative pathway. Thus, in PNH patients,any complement activation events that lead to C3b deposition on the RBCsurface can trigger subsequent amplification and pathological hemolysis(intravascular and/or extravascular) and precipitate a hemolytic crisis.A clear mechanistic understanding of the molecular events triggeringhemolytic crisis in PNH patients has remained elusive. Because nocomplement initiating event is overtly evident in PNH patientsundergoing a hemolytic crisis, the prevailing view is that complementactivation in PNH may occur spontaneously owing to low level “tick-over”activation of the alternative pathway, which is subsequently magnifiedby inappropriate control of terminal complement activation due to lackof CD55 and CD59.

However, it is important to note that in its natural history, PNHusually develops or is exacerbated after certain events, such as aninfection or an injury (Risitano, Biologics 2:205-222 (2008)), whichhave been shown to trigger complement activation. This complementactivation response is not dependent on prior immunity of the hosttowards the inciting pathogen, and hence likely does not involve theclassical pathway. Rather, it appears that this complement activationresponse is initiated by lectin binding to foreign or “altered self”carbohydrate patterns expressed on the surface of microbial agents ordamaged host tissue. Thus, the events precipitating hemolytic crisis inPNH are tightly linked to complement activation initiated via lectins.This makes it very likely that lectin activation pathways provide theinitiating trigger that ultimately leads to hemolysis in PNH patients.

Using well-defined pathogens that activate complement via lectins asexperimental models to dissect the activation cascades at the molecularlevel, we demonstrate that, depending on the inciting microbe,complement activation can be initiated by either LEA-2 or LEA-1, leadingto opsonization and/or lysis. This same principle of dual responses(i.e., opsonization and/or lysis) to lectin initiation events willlikely also apply to other types of infectious agents, or to complementactivation by lectins following tissue injury to the host, or otherlectin-driven complement activation events that may precipitate PNH. Onthe basis of this duality in the lectin pathway, we infer that LEA-2-and/or LEA-1-initiated complement activation in PNH patients promotesopsonization and/or lysis of RBCs with C3b and subsequent extravascularand intravascular hemolysis. Therefore, in the setting of PNH,inhibition of both LEA-1 and LEA-2 would be expected to address bothintravascular and extravascular hemolysis, providing a significantadvantage over the C5 inhibitor eculizumab.

It has been determined that exposure to S. pneumoniae preferentiallytriggers lectin-dependent activation of LEA-2, which leads toopsonization of this microbe with C3b. Since S. pneumonia is resistantto MAC-mediated lysis, its clearance from circulation occurs throughopsonisation with C3b. This opsonization and subsequent removal fromcirculation is LEA-2-dependent, as indicated by compromised bacterialcontrol in MASP-2-deficient mice and in mice treated with MASP-2monoclonal antibodies (PLOS Pathog., 8: e1002793. (2012)).

In exploring the role of LEA-2 in the innate host responses to microbialagents, we tested additional pathogens. A dramatically different outcomewas observed when Neisseria meningitidis was studied as a modelorganism. N. meningitidis also activates complement via lectins, andcomplement activation is required for containment of N. meningitidisinfections in the naive host. However, LEA-2 plays no host protectivefunctional role in this response: As shown in FIGS. 6 and 7, blockade ofLEA-2 through genetic ablation of MASP-2 does not reduce survivalfollowing infection with N. meningitidis. To the contrary, LEA-2blockade by MASP-2 ablation significantly improved survival (FIGS. 6 and7) as well as illness scores (FIG. 9) in these studies. LEA-2 blockadeby administration of MASP-2 antibody yielded the same result (FIG. 10),eliminating secondary or compensatory effects in the knockout-mousestrain as a possible cause. These favorable outcomes in LEA-2-ablatedanimals were associated with a more rapid elimination of N. meningitidisfrom the blood (FIG. 8). Also, as described herein, incubation of N.meningitidis with normal human serum killed N. meningitidis (FIG. 11).Addition of a functional monoclonal antibody specific for human MASP-2that blocks LEA-2, but not administration of an isotype controlmonoclonal antibody, may enhance this killing response. Yet, thisprocess depends on lectins and at least a partially functionalcomplement system, as MBL-deficient human serum or heat-inactivatedhuman serum was unable to kill N. meningitidis (FIG. 11). Collectively,these novel findings suggest that N. meningitidis infections in thepresence of a functional complement system are controlled by alectin-dependent but LEA-2-independent pathway of complement activation.

The hypothesis that LEA-1 may be the complement pathway responsible forlectin-dependent killing of N. meningitidis was tested using a serumspecimen from a 3MC patient. This patient was homozygous for a nonsensemutation in exon 12 of the MASP-1/3 gene. As a result, this patientlacked a functional MASP-3 protein, but was otherwise complementsufficient (exon 12 is specific for the MASP-3 transcript; the mutationhas no effect on MASP-1 function or expression levels) (see Nat Genet43(3):197-203 (2011)). Normal human serum efficiently kills N.meningitidis but heat-inactivated serum deficient in MBL (one of therecognition molecules for the Lectin pathway) and MASP-3-deficient serumwere unable to kill N. meningitidis (FIG. 12). Thus, LEA-1 appears tomediate N. meningitidis killing. This finding was confirmed using serumsamples from knockout mouse strains. While complement containing normalmouse serum readily killed N. meningitidis MBL-deficient orMASP-1/3-deficient mouse serum was as ineffective as heat-inactivatedserum that lacks functional complement (FIG. 13). Conversely,MASP-2-deficient serum exhibited efficient killing of N. meningitidis.

These findings provide evidence for a hitherto unknown duality in thelectin pathway by revealing the existence of separate LEA-2 and LEA-1pathways of lectin-dependent complement activation. In the examplesdetailed above, LEA-2 and LEA-1 are non-redundant and mediate distinct,functional outcomes. The data suggest that certain types of lectinpathway activators (including, but not limited to S. pneumonia)preferentially initiate complement activation via LEA-2 leading toopsonization, while others (exemplified by N. meningitidis)preferentially initiate complement activation via LEA-1 and promotecytolytic processes. The data do not, however, necessarily limit LEA-2to opsonization and LEA-1 to cytolytic processes, as both pathways inother settings can mediate opsonization and/or lysis.

In the context of lectin-dependent complement activation by N.meningitidis LEA-2 and LEA-1 arms appear to compete with each other, asblockade of LEA-2 enhanced LEA-1-dependent lytic destruction of theorganism in vitro (FIG. 13). As detailed above, this finding can beexplained by the increased likelihood of lectin MASP-1 complexesresiding in close proximity to lectin MASP-3 complexes in the absence ofMASP-2, which will enhance LEA-1 activation and thus promote moreeffective lysis of N. meningitides. Because lysis of N. meningitidis isthe main protective mechanism in the naïve host, blockade of LEA-2 invivo increases N. meningitidis clearance and leads to enhanced killing.

While the examples discussed above illustrate opposing effects of LEA-2and LEA-1 with respect to outcomes following infection with N.meningitidis there may be other settings where both LEA-2 and LEA-1 maysynergize to produce a certain outcome. As detailed below, in othersituations of pathological complement activation via lectins such asthose present in PNH, LEA-2- and LEA-1-driven complement activation maycooperate in a synergistic manner to contribute to the overall pathologyof PNH. In addition, as described herein, MASP-3 also contributes to thelectin-independent conversion of factor B and factor D, which can occurin the absence of Ca++, commonly leading to the conversion of C3bB toC3bBb and of pro-factor D to factor D, which may further contribute tothe pathology of PNH.

Biology and Expected Functional Activity in PNH

This section describes the inhibitory effects of LEA-2 and LEA-1blockade on hemolysis in an in vitro model of PNH. The findings supportthe utility of LEA-2-blocking agents (including, but not limited to,antibodies that bind to and block the function of MASP-2) andLEA-1-blocking agents (including, but not limited to, antibodies thatbind to and block the function of MASP-1-mediated activation of MASP-3,MASP-3, or both) to treat subjects suffering from one or more aspects ofPNH, and also the use of inhibitors of LEA-2 and/or LEA-1, and/orMASP-3-dependent, lectin-independent complement activation (includingMASP-2 inhibitors, MASP-3 inhibitors, and dual- or bispecificMASP-2/MASP-3 or MASP-1/MASP-2 inhibitors, and pan-specificMASP-1/MASP-2/MASP-3 inhibitors) to ameliorate the effects ofC3-fragment-mediated extravascular hemolysis in PNH patients undergoingtherapy with a C5-inhibitor such as eculizumab.

MASP-2 Inhibitors to Block Opsonization and Extravascular Hemolysis ofPNH RBCs Through the Reticuloendothelial System

As detailed above, PNH patients become anemic owing to two distinctmechanisms of RBC clearance from circulation: intravascular hemolysisvia activation of the membrane attack complex (MAC), and extravascularhemolysis following opsonization with C3b and subsequent clearancefollowing complement receptor binding and uptake by thereticuloendothelial system. The intravascular hemolysis is largelyprevented when a patient is treated with eculizumab. Because eculizumabblocks the terminal lytic effector mechanism that occurs downstream ofboth the complement-initiating activation event as well as the ensuingopsonization, eculizumab does not block extravascular hemolysis(Risitano A. M. et al., Blood 113:4094-100 (2009)). Instead, RBCs thatwould have undergone hemolysis in untreated PNH patients now canaccumulate activated C3b proteins on their surface, which augmentsuptake by the reticuloendothelial system and enhances theirextravascular hemolysis. Thus, eculizumab treatment effectively divertsRBC disposition from intravascular hemolysis to potential extravascularhemolysis. As a result, some eculizumab-treated PNH patients remainanemic. It follows that agents that block complement activation upstreamand prevent the opsonization of PNH RBCs may be particularly suitable toblock the extravascular hemolysis occasionally seen with eculizumab.

The microbial data presented here suggest that LEA-2 is often thedominant route for lectin-dependent opsonization. Furthermore, whenlectin-dependent opsonization (measured as C3b deposition) was assessedon three prototypic lectin activation surfaces (mannan, FIG. 17A;zymosan, FIG. 17B, and S. pneumonia ; FIG. 17C), LEA-2 appears to be thedominant route for lectin-dependent opsonization under physiologicconditions (i.e., in the presence of Ca⁺⁺ wherein all complementpathways are operational). Under these experimental conditions,MASP-2-deficient serum (which lacks LEA-2) is substantially lesseffective in opsonizing the test surfaces than WT serum.MASP-1/3-deficient serum (which lacks LEA-1) is also compromised, thoughthis effect is much less pronounced as compared to serum lacking LEA-2.The relative magnitude of the contributions of LEA-2 and LEA-1 tolectin-driven opsonization is further illustrated in FIGS. 18A-18C.While the alternative pathway of complement has been reported to supportopsonization of lectin activating surfaces in the absence of lectinpathway or classical pathway (Selander et al., J Clin Invest116(5):1425-1434 (2006)), the alternative pathway in isolation (measuredunder Ca⁺⁺-free assay conditions) appears substantially less effectivethan the LEA-2- and LEA-1-initiated processes described herein. Byextrapolation, these data suggest that opsonization of PNH RBCs may alsobe preferentially initiated by LEA-2 and, to a lesser extent, by LEA-1(possibly amplified by the alternative pathway amplification loop),rather than the result of lectin-independent alternative pathwayactivation. Therefore, LEA-2 inhibitors may be expected to be mosteffective at limiting opsonization and preventing extravascularhemolysis in PNH. However, recognition of the fact that lectins otherthan MBL, such as ficolins, bind to non-carbohydrate structures such asacetylated proteins, and that MASP-3 preferentially associates withH-ficolin (Skjoedt et al., Immunobiol. 215:921-931, 2010), leaves openthe possibility of a significant role for LEA-1 in PNH-associated RBCopsonization as well. Therefore, LEA-1 inhibitors are expected to haveadditional anti-opsonization effects, and the combination of LEA-1 andLEA-2 inhibitors is expected to be optimal and mediate the most robusttreatment benefit in limiting opsonization and extravascular hemolysisin PNH patients. Thus, LEA-2 and LEA-1 act additively or synergisticallyto promote opsonization, and a crossreactive or bispecific LEA-1/LEA-2inhibitor is expected to be most effective at blocking opsonization andextravascular hemolysis in PNH.

Role of MASP-3 Inhibitors in PNH

Using an in vitro model of PNH, we demonstrated that complementactivation and the resulting hemolysis in PNH are indeed initiated byLEA-2 and/or LEA-1 activation, and that it is not an independentfunction of the alternative pathway. These studies usedmannan-sensitized RBCs of various mouse stains, including RBCs fromCrry-deficient mice (an important negative regulator of the terminalcomplement pathway in mice) as well as RBCs from CD55/CD59-deficientmice, which lack the same complement regulators that are absent in PNHpatients). When mannan-sensitized Crry-deficient RBCs were exposed tocomplement-sufficient human serum, the RBCs effectively hemolysed at aserum concentration of 3% (FIGS. 19 and 20) while complement-deficientserum (HI: heat-inactivated) was not hemolytic. Remarkably,complement-sufficient serum where LEA-2 was blocked by addition ofMASP-2 antibody had reduced hemolytic activity, and 6% serum was neededfor effective hemolysis. Similar observations were made whenCD55/CD59-deficient RBCs were tested (FIG. 22). Complement-sufficienthuman serum supplemented with MASP-2 monoclonal antibody (i.e., serumwhere LEA-2 is suppressed) was about two-fold less effective thanuntreated serum in supporting hemolysis. Furthermore, higherconcentrations of LEA-2-blocked serum (i.e., treated with antiMASP-2monoclonal antibody) were needed to promote effective hemolysis ofuntreated WT RBCs compared to untreated serum (FIG. 21).

Even more surprisingly, serum from a 3MC patient homozygous for adysfunctional MASP-3 protein (and hence lacking LEA-1) was completelyunable to hemolyze mannan-sensitized Crry-deficient RBCs (FIG. 20 andFIG. 21). A similar outcome was observed when unsensitized normal RBCswere used: As shown in FIG. 21, LEA-1-defective serum isolated from a3MC patient was completely ineffective at mediating hemolysis.Collectively, these data indicate that whereas LEA-2 contributessignificantly to the intravascular hemolysis response, LEA-1 is thepredominant complement-initiating pathway leading to hemolysis. Thus,while LEA-2 blocking agents are expected to significantly reduceintravascular hemolysis of RBCs in PNH patients, LEA-1 blocking agentsare expected to have a more profound effect and largely eliminatecomplement-driven hemolysis.

It should be noted that the serum of the LEA-1-deficient 3MC patientused in this study possessed a diminished but functional alternativepathway when tested under conventional alternative pathway assayconditions (FIG. 15). This finding suggests that LEA-1 makes a greatercontribution to hemolysis than alternative pathway activity asconventionally defined in this experimental setting of PNH. Byinference, it is implied that LEA-1-blocking agents will be at least aseffective as agents blocking other aspects of the alternative pathway inpreventing or treating intravascular hemolysis in PNH patients.

Role of MASP-2 Inhibitors in PNH

The data presented herein suggest the following pathogenic mechanismsfor anemia in PNH: intravascular hemolysis due to unregulated activationof terminal complement components and lysis of RBC by formation of MAC,which is initiated predominantly, though not exclusively, by LEA-1, andextravascular hemolysis caused by opsonization of RBCs by C3b, whichappears to be initiated predominately by LEA-2. While a discernible rolefor LEA-2 in initiating complement activation and promoting MACformation and hemolysis is apparent, this process appears substantiallyless effective than LEA-1-initiated complement activation leading tohemolysis. Thus, LEA-2-blocking agents are expected to significantlyreduce intravascular hemolysis in PNH patients, though this therapeuticactivity is expected to be only partial. By comparison, a moresubstantial reduction in intravascular hemolysis in PNH patients isexpected for LEA-1-blocking agents.

Extravascular hemolysis, a less dramatic, yet equally importantmechanism of RBC destruction that leads to anemia in PNH, is primarilythe result of opsonization by C3b, which appears to be predominantlymediated by LEA-2. Thus, LEA-2-blocking agents may be expected topreferentially block RBC opsonization and the ensuing extravascularhemolysis in PNH. This unique therapeutic activity of LEA-2-blockingagents is expected to provide a significant treatment benefit to all PNHpatients as no treatment currently exists for those PNH patients whoexperience this pathogenic process.

LEA-2 Onhibitors as Adjunct Treatment to LEA-1 Inhibitors or TerminalComplement Blocking Agents

The data presented herein detail two pathogenic mechanisms for RBCclearance and anemia in PNH which can be targeted, separately or incombination, by distinct classes of therapeutic agents: theintravascular hemolysis initiated predominantly, though not exclusively,by LEA-1 and thus expected to be effectively prevented by aLEA-1-blocking agent, and extravascular hemolysis due to C3bopsonization driven predominantly by LEA-2, and thus effectivelyprevented by a LEA-2-blocking agent.

It is well documented that both intravascular and extravascularmechanisms of hemolysis lead to anemia in PNH patients (Risitano et al.,Blood 113:4094-4100 (2009)). Therefore, it is expected that aLEA-1-blocking agent that prevents intravascular hemolysis incombination with a LEA-2 blocking agent that primarily preventsextravascular hemolysis will be more effective than either agent alonein preventing the anemia that develops in PNH patients. In fact, thecombination of LEA-1- and LEA-2-blocking agents is expected to preventall relevant mechanisms of complement initiation in PNH and thus blockall symptoms of anemia in PNH.

It is also known that C5-blocking agents (such as eculizumab)effectively block intravascular hemolysis but do not interfere withopsonization. This leaves some anti-C5-treated PNH patients withsubstantial residual anemia due to extravascular hemolysis mediated byLEA-2 that remains untreated. Therefore, it is expected that aC5-blocking agent (such as eculizumab) that prevents intravascularhemolysis in combination with a LEA-2 blocking agent that reducesextravascular hemolysis will be more effective than either agent alonein preventing the anemia that develops in PNH patients.

Other agents that block the terminal amplification loop of thecomplement system leading to C5 activation and MAC deposition(including, but not limited to agents that block properdin, factor B orfactor D or enhance the inhibitory activity of factor I, factor H orother complement inhibitory factors) are also expected to inhibitintravascular hemolysis. However, these agents are not expected tointerfere with LEA-2-mediated opsonization in PNH patients. This leavessome PNH patients treated with such agents with substantial residualanemia due to extravascular hemolysis mediated by LEA-2 that remainsuntreated. Therefore, it is expected that treatment with such agentsthat prevent intravascular hemolysis in combination with aLEA-2-blocking agent that minimizes extravascular hemolysis will be moreeffective than either agent alone in preventing the anemia that developsin PNH patients. In fact, the combination of such agents and a LEA-2blocking agent is expected to prevent all relevant mechanisms of RBCdestruction in PNH and thus block all symptoms of anemia in PNH.

Use of LEA-1 and LEA-2 Multiple, Bispecific or Pan-Specific Antibodiesto Treat PNH

As detailed above, the use of a combination of pharmacologic agents thatindividually block LEA-1 and LEA-2, and thus in combination block allcomplement activation events that mediate the intravascular as well asthe extravascular hemolysis, is expected to provide the best clinicaloutcome for PNH patients. This outcome can be achieved for example, byco-administration of an antibody that has LEA-1-blocking activitytogether with an antibody that has LEA-2-blocking activity. In someembodiments, LEA-1- and LEA-2-blocking activities are combined into asingle molecular entity, and that such entity with combined LEA-1- andLEA-2-blocking activity will effectively block intravascular as well asthe extravascular hemolysis and prevent anemia in PNH. Such an entitymay comprise or consist of a bispecific antibody where oneantigen-combining site specifically recognizes MASP-1 and blocks LEA-1and diminishes LEA-2 and the second antigen-combining site specificallyrecognizes MASP-2 and further blocks LEA-2. Alternatively, such anentity may consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes MASP-3 and thus blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Such an entity may optimally consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1 anddiminishes LEA-2 while the second antigen-combining site specificallyrecognized MASP-2 and further blocks LEA-2. Based on the similarities inthe overall protein sequence and architecture, it can also be envisionedthat a conventional antibody with two identical binding sites can bedeveloped that specifically binds to MASP-1 and to MASP-2 and to MASP-3in a functional manner, thus achieving functional blockade of LEA-1 andLEA-2. Such an antibody with pan-MASP inhibitory activity is expected toblock both the intravascular as well as the extravascular hemolysis andthus effectively treat the anemia in PNH patients.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as PNH.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developing PNHcomprising an effective amount of a high affinity monoclonal antibody orantigen binding fragment thereof as disclosed herein that binds to humanMASP-3 and inhibits alternative pathway complement activation to treator reduce the risk of PNH in the subject.

In one embodiment, the present invention provides a method for treatinga subject suffering from, or at risk for developing paroxysmal nocturnalhemoglobinuria (PNH), comprising administering to the subject apharmaceutical composition comprising an effective amount of amonoclonal antibody or antigen binding fragment thereof as disclosedherein that binds to human MASP-3 and inhibits alternative pathwaycomplement activation to treat or reduce the risk of PNH in the subject,such as, wherein said antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising (i) VHCDR1comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ IDNO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a light chainvariable region comprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ IDNO:257, SEQ ID NO:258 or SEQ ID NO:259, (ii) VLCDR2 comprising SEQ IDNO:144 and (iii) VLCDR3 comprising SEQ ID NO:161. In some embodiments,the pharmaceutical composition increases the survival of red blood cellsin the subject suffering from PNH. In some embodiments, wherein thesubject suffering from or at risk for developing PNH exhibits one ormore symptoms selected from the group consisting of (i) below normallevels of hemoglobin, (ii) below normal levels of platelets; (iii) abovenormal levels of reticulocytes, and (iv) above normal levels ofbilirubin. In some embodiments, the pharmaceutical composition isadministered systemically (e.g., subcutaneously, intra-muscularly,intravenously, intra-arterially or as an inhalant) to a subjectsuffering from, or at risk for developing PNH. In some embodiments, thesubject suffering from or at risk for PNH has previously undergone, oris currently undergoing treatment with a terminal complement inhibitorthat inhibits cleavage of complement protein C5. In some embodiments,the method further comprises administering to the subject a terminalcomplement inhibitor that inhibits cleavage of complement protein C5. Insome embodiments, the terminal complement inhibitor is a humanizedanti-C5 antibody or antigen-binding fragment thereof. In someembodiments, the terminal complement inhibitor is eculizumab.

B. The Role of MASP-3 In Age-related Macular Degeneration andTherapeutic Methods Using MASP-3 Inhibitory Antibodies, Optionally inCombination with And MASP-2 Inhibitory Agents

Age related macular degeneration (AMD) is the leading cause of visualimpairment and blindness in the elderly and accounts for up to 50% ofcases of blindness in developed countries. The prevalence of AMD isaround 3% in adults and increases with age such that almost two-thirdsof the population over 80 years of age will have some signs. It isestimated that over 1.75 million individuals in the United States haveadvanced AMD and the prevalence is increasing as the population ages andis expected to reach almost 3 million by 2020 (Friedman, D. S., et al.,Arch. Ophthalmol. 122:564-572, 2004). AMD is an abnormality of theretinal pigment epithelium (RPE) that results in degeneration of thephotoreceptors of the overlying central retina, or macula, and loss ofcentral vision. Early and intermediate forms of AMD are characterized byprogressive deposits of drusen, a yellowish material containing lipid,protein, lipoprotein, and cellular debris, in the subretinal spaceadjacent to the RPE, along with pigmentary irregularities in the retina.Advanced AMD consists of two clinical subtypes: non-neovasculargeographic atrophic (‘dry’) AMD and neovascular exudative (‘wet’) AMD.Although dry AMD accounts for 80-90% of advanced AMD, the majority ofsudden and severe vision loss occurs in patients with wet AMD. It is notknown whether the two types of AMD represent differing phenotypesarising from similar pathologies or two distinct conditions. Currentlyno therapy has been approved by the United States Food and DrugAdministration (FDA) to treat dry AMD. FDA-approved treatment optionsfor wet AMD include intravitreal injections of anti-angiogenic drugs(ranibizumab, pegaptanib sodium, aflibercept), laser therapy,photodynamic laser therapy, and implantable telescope.

The etiology and pathophysiology of AMD are complex and incompletelyunderstood. Several lines of evidence support the role of dysregulationof the complement system in the pathogenesis of AMD. Gene associationstudies have identified multiple genetic loci associated with AMD,including genes coding for a range of complement proteins, factors, andregulators. The strongest association is with polymorphisms in thecomplement factor H (CFH) gene, with the Y402H variant homozygoteshaving approximately 6-fold and heterozygotes approximately 2.5-foldincreased risk for developing AMD compared to the non-risk genotype(Khandhadia, S., et al., Immunobiol. 217:127-146, 2012). Mutations inother complement pathway encoding genes have also been associated withincreased or decreased risk of AMD, including complement factor B (CFB),C2, C3, factor I, and CFH-related proteins 1 and 3 (Khandhadia et al.).Immunohistochemical and proteomic studies in donor eyes from AMDpatients showed that proteins of the complement cascade to be increasedand localized in drusen (Issa, P. C., et al., Graefes. Arch. Clin. Exp.Ophthalmol. 249:163-174, 2011). Furthermore, AMD patients have increasedsystemic complement activation as measured in peripheral blood (Issa etal., 2011, supra).

The alternative pathway of complement appears to be more relevant thanthe classical pathway in the pathogenesis of AMD. C1q, the essentialrecognition component for activation of the classical pathway, was notdetected in drusen by immunohistochemical analyses (Mullins et al.,FASEB J. 14:835 846, 2000; Johnson et al., Exp. Eye Res. 70:441 449,2000). Genetic association studies have implicated CFH and CFB genes.These proteins are involved in the alternative pathway amplificationloop, with CFH being a fluid phase inhibitor and CFB being an activatingprotease component of the alternative pathway. The Y402H variant of CFHaffects interaction with ligand binding, including binding withC-reactive protein, heparin, M protein, and glycosaminoglycans. Thisaltered binding to ligands may reduce binding to cell surfaces, which inturn may lead to reduced factor I mediated degradation of C3b activationfragment and impaired regulation of the alternative C3 convertase,resulting in over activation of the alternative pathway (Khandhadia etal., 2012, supra). Variations in the CFB gene are associated with aprotective effect for the development of AMD. A functional variant fB32Qhad 4 times less binding affinity to C3b than the risk variant fB32R,resulting in a reduction in C3 convertase formation (Montes, T. et al.,Proc. Natl. Acad. Sci. U.S.A. 106:4366-4371, 2009).

Complement-Initiating Mechanisms in AMD

The human genetic linkage studies discussed above suggest an importantrole for the complement system in AMD pathogenesis. Furthermore,complement activation products are abundantly present in drusen (Issa,P. C., et al., Graefes. Arch. Clin. Exp. Ophthalmol. 249:163-174, 2011),a hallmark pathologic lesion in both wet and dry AMD. However, thenature of the events initiating complement activation, and thecomplement activation pathway(s) involved remain incompletelyunderstood.

It is important to note that drusen deposits are composed of cellulardebris and oxidative waste products originating from the retina thataccumulate beneath the RPE as the eye ages. In addition, oxidativestress appears to play an important role (Cai et al; Front Biosci.,17:1976-95, 2012), and has been shown to cause complement activation inRPE (J Biol Chem., 284(25):16939-47, 2009). It is widely appreciatedthat both oxidative stress and cellular or tissue injury activate thecomplement system lectins. For example, Collard et al. have demonstratedthat endothelial cells exposed to oxidative stress trigger abundantcomplement deposition mediated by lectins (Collard C D et al., MolImmunol., 36(13-14):941-8, 1999; Collard C. D. et al., Am J Pathol.,156(5):1549-56, 2000), and that blockade of lectin binding andlectin-dependent complement activation improves outcomes in experimentalmodels of oxidative stress injury (Collard C. D. et al., Am JPathol.,156(5):1549-56, 2000). Thus, it appears likely that oxidativewaste products present in drusen also activate complement via thelectins. By inference, lectin-dependent complement activation may play apivotal role in AMD pathogenesis.

The role of the complement system has been evaluated in mouse models ofAMD. In the light-damage mouse model, an experimental model foroxidative stress-mediated photoreceptor degeneration, knockout mice withan elimination of the classical pathway (C1qa−/−on a C57BL/6 background)had the same sensitivity to light damage compared to wild-typelittermates, whereas elimination of complement factor D of thealternative pathway (CFD−/−) resulted in protection from light damage(Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 48:5282-5289, 2007).In a mouse model of choroidal neovascularization (CNV) induced by laserphotocoagulation of the Bruch membrane, knockout mice without complementFactor B (CFB−/−) were protected against CNV compared with wild-typemice (Rohrer, B. et al., Invest. Ophthalmol. Vis. Sci. 50:3056-3064,2009). In the same model, intravenous administration of a recombinantform of complement Factor H targeted to sites of complement activation(CR2-fH) reduced the extent of CNV. This protective effect was observedwhether CR2-fH was administered at the time of laser injury ortherapeutically (after laser injury). A human therapeutic version ofCR2-fH (TT30) was also efficacious in the murine CNV model (Rohrer, B.et al. J. Ocul. Pharmacol. Ther.,28:402-409, 2012). Because fB isactivated by LEA-1, and because MASP-1 and MASP-3 contribute to thematuration of factor D, these findings imply that LEA-1 inhibitors mayhave therapeutic benefit in AMD patients. In addition, recent resultsreported from a Phase 2 study have shown that monthly intravitrealinjection with Lampalizumab (previously referred to as FCFD4514S andanti-factor D, which is an antigen-binding fragment of a humanizedmonoclonal antibody directed against Factor D) reduced geographicatrophy area progression in patients with geographic atrophy secondaryto AMD (Yaspan B. L. et al., Sci Transl. Med. 9, Issue 395, Jun. 21,2017).

Initial experimental studies in a rodent model of AMD usingMBL-deficient mice did not support a critical role for the lectinpathway in pathogenic complement activation (Rohrer et al., Mol Immunol.48:e1-8, 2011). However, MBL is only one of several lectins, and lectinsother than MBL may trigger complement activation in AMD. Indeed, ourprevious work has shown that MASP-2, the rate-limiting serine proteasethat is critically required for lectin pathway function, plays acritical role in AMD. As described in U.S. Pat. No. 7,919,094 (assignedto Omeros Corporation), incorporated herein by reference,MASP-2-deficient mice and mice treated with MASP-2 antibody wereprotected in a mouse model of laser-induced CNV, a validated preclinicalmodel of wet AMD (Ryan et al., Tr Am Opth Soc LXXVII:707-745, 1979).Thus, inhibitors of LEA-2 are expected to effectively prevent CNV andimprove outcomes in AMD patients.

Thus, in view of the above, LEA-1 and LEA-2 inhibitors are expected tohave independent therapeutic benefit in AMD. In addition, LEA-1 andLEA-2 inhibitors used together may achieve additional treatment benefitcompared to either agent alone, or may provide effective treatment for awider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibitionmay be accomplished by co-administration of a LEA-1-blocking agent and aLEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function maybe encompassed in a single molecular entity, such as a bispecificantibody composed of MASP-1/3 and a MASP-2-specific binding site, or adual specificity antibody where each binding site can bind to and blockMASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationto treat age-related macular degeneration (wet and dry forms) byadministering a composition comprising a therapeutically effectiveamount of a MASP 1 inhibitory agent, a MASP 3 inhibitory agent, or acombination of a MASP ⅓ inhibitory agent, in a pharmaceutical carrier toa subject suffering from such a condition. The MASP 1, MASP 3, or MASP1/3 inhibitory composition may be administered locally to the eye, suchas by irrigation, intravitreal administration, or application of thecomposition in the form of a gel, salve or drops. Alternately, the MASP1, MASP 3, or MASP 1/3 inhibitory agent may be administered to thesubject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

In one embodiment, the method according to this aspect of the inventionfurther comprises inhibiting LEA-2-dependent complement activation in asubject suffering from age-related macular degeneration, comprisingadministering a therapeutically effective amount of a MASP-2 inhibitoryagent and a MASP-1, MASP-3 or MASP1/3 inhibitory agent to the subject inneed thereof. As detailed above, the use of a combination ofpharmacologic agents that individually block LEA-1 and LEA-2 is expectedto provide an improved therapeutic outcome in AMD patients as comparedto the inhibition of LEA-1 alone. This outcome can be achieved forexample, by co-administration of an antibody that has LEA-1-blockingactivity together with an antibody that has LEA-2-blocking activity. Insome embodiments, LEA-1- and LEA-2-blocking activities are combined intoa single molecular entity, and that such entity with combined LEA-1- andLEA-2-blocking activity. Such an entity may comprise or consist of abispecific antibody where one antigen-combining site specificallyrecognizes MASP-1 and blocks LEA-1 and the second antigen-combining sitespecifically recognizes MASP-2 and blocks LEA-2. Alternatively, such anentity may consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes MASP-3 and thus blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Such an entity may optimally consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1while the second antigen-combining site specifically recognized MASP-2and blocks LEA-2.

The MASP 2 inhibitory composition may be administered locally to theeye, such as by irrigation, intravitreal injection or topicalapplication of the composition in the form of a gel, salve or drops.Alternately, the MASP 2 inhibitory agent may be administered to thesubject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and optional MASP 2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treatment of AMD.Alternatively, the composition may be administered at periodic intervalssuch as daily, biweekly, weekly, every other week, monthly or bimonthlyover an extended period of time for treatment of AMD.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as AMD.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developing AMDcomprising an effective amount of a high affinity monoclonal antibody orantigen binding fragment thereof as disclosed herein that binds to humanMASP-3 and inhibits alternative pathway complement activation to treator reduce the risk of AMD in the subject. In one embodiment, the presentinvention provides a method for treating a subject suffering from, or atrisk for developing AMD comprising administering to the subject apharmaceutical composition comprising an effective amount of amonoclonal antibody or antigen binding fragment thereof as disclosedherein that binds to human MASP-3 and inhibits alternative pathwaycomplement activation to treat or reduce the risk of AMD in the subject,such as, for example, wherein said antibody or antigen binding fragmentthereof comprises (a) a heavy chain variable region comprising (i)VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprising SEQ ID NO:86 orSEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88; and (b) a lightchain variable region comprising (i) VLCDR1 comprising SEQ ID NO:142,SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259, (ii) VLCDR2 comprisingSEQ ID NO:144 and (iii) VLCDR3 comprising SEQ ID NO:161.

C. The Role of MASP-3 in Ischemia Reperfusion Injury and TherapeuticMethods Using MASP-3 Inhibitory Antibodies, Optionally in Combinationwith MASP-2 Inhibitory Agents

Tissue ischemia is the basis for a wide spectrum of clinical disorders.Although timely restoration of blood flow is essential to preservationof ischemic tissue, it has long been recognized that reperfusion, whichcan occur either spontaneously or through therapeutic intervention, maylead to additional tissue injury, a phenomenon that has been termedischemia reperfusion (I/R) injury (Eltzschig, H. K. and Tobias, E., Nat.Med. 17:1391-1401, 2011). I/R injury may affect single organs, such asthe heart (acute coronary syndrome), kidney (acute kidney injury),intestine (intestinal I/R), and brain (stroke). I/R injury may alsoaffect multiple organs, such as following major trauma and resuscitation(multiple organ failure), circulatory arrest (hypoxic brain injury,acute kidney injury), peripheral vascular disease, and sickle celldisease (acute chest syndrome, acute kidney injury). Major surgery maybe associated with I/R injury, including cardiac surgery (acute heartfailure after cardiopulmonary bypass), thoracic surgery (acute lunginjury), peripheral vascular surgery (compartment syndrome), vascularsurgery (acute kidney injury), and solid organ transplantation (acutegraft failure). Currently there are no specific therapies that targetI/R injury and there is a need for effective treatments in order tomaximize the salvage of tissue in the ischemic zone and improvefunctional outcome in these common settings.

The pathophysiology of I/R injury is complex and characterized by arobust inflammatory response following reperfusion. Activation of thecomplement system has been implicated as an important component of I/Rinjury and inhibition of complement activity has been efficacious in avariety of animal models (Diepenhorst, G. M. P. et al., Ann. Surg.249:889-899, 2009). The relative importance of the classical, lectin,and alternative pathways in I/R injury is largely unsettled and maydiffer depending on the organs affected. Recently the availability ofknockout mice deficient in specific complement proteins andpathway-specific inhibitors has generated data that implicate the lectinand alternative pathways in I/R injury.

The role of the alternative pathway in gastrointestinal I/R injury wasinvestigated using factor D-deficient (−/−) and heterozygotus (+/−) mice(Stahl, G. L., et al. Am. J. Pathol. 162:449-455, 2003). Followingtransient gastrointestinal ischemia, intestinal and pulmonary injurywere reduced but not prevented in factor D-deficient mice compared withheterozygotus mice, and addition of human factor D to Factor D (−/−)mice restored I/R injury. The same model was evaluated in C1q-deficientand MBL-A/C-deficient mice and the results showed that gastrointestinalI/R injury was independent of C1q and classical pathway activation, butthat MBL and lectin pathway activation was required for intestinalinjury (Hart, M. L., et al. J. Immunol. 174:6373-6380, 2005).Conversely, the C1q recognition molecule of the classical pathway wasresponsible for pulmonary injury after intestinal I/R (Hart, M. L., etal. J. Immunol. 174:6373-6380, 2005). One hypothesis is that activationof complement during I/R injury occurs through natural IgM binding toself-antigens present on the surface of ischemic (but not normal)tissue, for example non-muscle myosin heavy chains type II. In a mousegastrointestinal I/R injury model, immunocomplexes from gut tissue wereevaluated for the presence of initiating factors in the classical (C1q),lectin (MBL), or alternative (Factor B) pathways (Lee, H., et al., Mol.Immunol. 47:972-981, 2010). The results showed that C1q and MBL weredetected whereas Factor B was not detected in these immunocomplexes,indicating involvement of the classical and lectin pathways but not thealternative pathway. In the same model, Factor B-deficient mice were notprotected from local tissue injury, providing additional support for thelack of involvement of the alternative pathway. The role of the lectinpathway in gastrointestinal I/R injury was directly evaluated inMASP-2-deficient mice and the results showed that gastrointestinalinjury was reduced in these mice compared with wide-type controls;treatment with MASP-2 monoclonal antibody was similarly protective(Schwaeble, W. J., et al., Proc. Natl. Acad. Sci. 108:7523-7528, 2011).Taken together, these results provide support for the involvement of thelectin pathway in gastrointestinal I/R injury, with conflicting dataregarding involvement of the alternative pathway.

In a mouse myocardial I/R injury model, a pathogenic role wasdemonstrated for the lectin pathway as MBL-deficient mice were protectedfrom myocardial injury whereas C1q-deficient and C2/fB-deficient micewere not (Walsh, M. C. et al., J. Immunol. 175:541-546, 2005).Protection from myocardial I/R injury was also observed inMASP-2-deficient mice (Schwaeble, W. J., et al., Proc. Natl. Acad. Sci.108:7523-7528, 2011). Treatment of rats in a myocardial I/R model withmonoclonal antibodies against rat MBL resulted in reduced post-ischemicreperfusion injury (Jordan, J. E., et al., Circulation 104:1413 18,2001). In a study of myocardial infarction patients treated withangioplasty, MBL deficiency was associated with reduced 90-day mortalitycompared to MBL-sufficient counterparts (M Trendelenburg et al., EurHeart J. 31:1181, 2010). Furthermore, myocardial infarction patientsthat develop cardiac dysfunction after angioplasty haveapproximately˜three-fold higher MBL levels compared to patients withfunctional recovery (Haahr-Pedersen S., et al., J Inv Cardiology, 21:13,2009). MBL antibodies also reduced complement deposition on endothelialcells in vitro after oxidative stress indicating a role for the lectinpathway in myocardial I/R injury (Collard, C. D., et al., Am. J. Pathol.156:1549 56, 2000). In a mouse heterotopic isograft heart transplantmodel of I/R injury, the role of the alternative pathway wasinvestigated using the pathway-specific fusion protein CR2-fH (Atkinson,C., et al., J. Immunol. 185:7007-7013, 2010). Systemic administration ofCR2-fH immediately posttransplantation resulted in a reduction inmyocardial I/R injury to an extent comparable to treatment withCR2-Crry, which inhibits all complement pathways, indicating that thealternative pathway is of key importance in this model.

In a mouse model of renal I/R injury, the alternative pathway wasimplicated as factor B-deficient mice were protected from a decline inrenal function and tubular injury, compared with wild-type mice(Thurman, J. M., et al., J. Immunol. 170:1517-1523, 2003). Treatmentwith an inhibitory monoclonal antibody to factor B prevented complementactivation and reduced murine renal I/R injury (Thurman, J. M., et al.,J. Am. Soc. Nephrol. 17:707-715, 2006). In a bilateral renal I/R injurymodel, MBL-A/C-deficient mice were protected from kidney damage comparedwith wild-type mice and recombinant human MBL reversed the protectiveeffect in MBL-A/C-deficient mice, implicating a role for MBL in thismodel (Moller-Kristensen, M., et al., Scand. J. Immunol. 61:426-434,2005). In a rat unilateral renal I/R injury model, inhibition of MBLwith a monoclonal antibody to MBL-A preserved renal function after I/R(van der Pol, P., et al., Am. J. Transplant. 12:877-887, 2010).Interestingly, the role of MBL in this model did not appear to involveactivation of the terminal complement components, as treatment with a C5antibody was ineffective in preventing renal injury. Rather, MBLappeared to have a direct toxic effect on tubular cells, as humanproximal tubular cells incubated with MBL in vitro internalized MBL withsubsequent cellular apoptosis. In a swine model of renal I/R, CastellanoG. et al., (Am J Pathol, 176(4):1648-59, 2010), tested a C1 inhibitor,which irreversibly inactivates C1r and C1s proteases in the classicalpathway and also MASP-1 and MASP-2 proteases in MBL complexes of thelectin pathway, and found that C1 inhibitor reduced complementdeposition in peritubular capillaries and glomerulus and reduced tubulardamage.

The alternative pathway appears to be involved in experimental traumaticbrain injury as factor B-deficient mice had reduced systemic complementactivation as measured by serum C5a levels and reduced posttraumaticneuronal cell death compared with wide-type mice (Leinhase, I., et al.,BMC Neurosci. 7:55-67, 2006). In human stroke, complement componentsC1q, C3c, and C4d were detected by immunohistochemical staining inischemic lesions, suggesting activation via the classical pathway(Pedersen, E. D., et al., Scand. J. Immunol. 69:555-562, 2009).Targeting of the classical pathway in animal models of cerebral ischemiahas yielded mixed results, with some studies demonstrating protectionwhile others showing no benefit (Arumugam, T. V., et al., Neuroscience158:1074-1089, 2009). Experimental and clinical studies have providedstrong evidence for lectin pathway involvement. In experimental strokemodels, deficiency of either MBL or MASP-2 results in reduced infarctsizes compared to wild-type mice (Cervera A, et al.; PLoS One3;5(2):e8433, 2010; Osthoff M. et al., PLoS One, 6(6):e21338, 2011).Furthermore, stroke patients with low levels of MBL have a betterprognosis compared to their MBL-sufficient counterpart (Osthoff M. etal., PLoS One, 6(6):e21338, 2011).

In a baboon model of cardiopulmonary bypass, treatment with a factor Dmonoclonal antibody inhibited systemic inflammation as measured byplasma levels of C3a, sC5b-9, and IL-6, and reduced myocardial tissueinjury, indicating involvement of the alternative pathway in this model(Undar, A., et al., Ann. Thorac. Surg. 74:355-362, 2002).

Thus, depending on the organ affected by I/R, all three pathways ofcomplement can contribute to pathogenesis and adverse outcomes. Based onthe experimental and clinical findings detailed above, LEA-2 inhibitorsare expected to be protective in most settings of I/R. Lectin-dependentactivation of LEA-1 may cause complement activation via the alternativepathway at least in some settings. In addition, LEA-2-initiatedcomplement activation may be further amplified by the alternativepathway amplification loop and thus exacerbate I/R-related tissueinjury. Thus, LEA-1 inhibitors are expected to provide additional orcomplementary treatment benefits in patients suffering from anischemia-related condition.

In view of the above, LEA-1 and LEA-2 inhibitors are expected to haveindependent therapeutic benefits in treating, preventing or reducing theseverity of ischemia-reperfusion related conditions. In addition, LEA-1and LEA-2 inhibitors used together may achieve additional treatmentbenefits compared to either agent alone. An optimally effectivetreatment for an I/R-related condition therefore comprises activepharmaceutical ingredients that, alone or in combination, block bothLEA-1 and LEA-2. Combined LEA-1 and LEA-2 inhibition may be accomplishedby co-administration of a LEA-1 blocking agent and a LEA-2 blockingagent. Preferentially, LEA-1 and LEA-2 inhibitory function may beencompassed in a single molecular entity, such as a bispecific antibodycomposed of MASP-1/3 and a MASP-2-specific binding site, or a dualspecificity antibody where each binding site can bind to and blockMASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing or reducing the severity of ischemiareperfusion injuries by administering a composition comprising atherapeutically effective amount of a LEA-1 inhibitory agent comprisinga MASP 1 inhibitory agent, a MASP 3 inhibitory agent, or a combinationof a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subjectexperiencing ischemic reperfusion. The MASP 1, MASP 3, or MASP 1/3inhibitory composition may be administered to the subject by intraarterial, intravenous, intracranial, intramuscular, subcutaneous, orother parenteral administration, and potentially orally for nonpeptidergic inhibitors, and most suitably by intra arterial orintravenous administration. Administration of the LEA-1 inhibitorycompositions of the present invention suitably commences immediatelyafter or as soon as possible after an ischemia reperfusion event. Ininstances where reperfusion occurs in a controlled environment (e.g.,following an aortic aneurism repair, organ transplant or reattachment ofsevered or traumatized limbs or digits), the LEA-1 inhibitory agent maybe administered prior to and/or during and/or after reperfusion.Administration may be repeated periodically as determined by a physicianfor optimal therapeutic effect.

In some embodiments, the methods are used to treat or prevent anischemia-reperfusion injury associated with at least one of aorticaneurysm repair, cardiopulmonary bypass, vascular reanastomosis inconnection with organ transplants and/or extremity/digit replantation,stroke, myocardial infarction, and hemodynamic resuscitation followingshock and/or surgical procedures.

In some embodiments, the methods are used to treat or prevent anischemia-reperfusion injury in a subject that is about to undergo, isundergoing, or has undergone an organ transplant. In some embodiments,the methods are used to treat or prevent an ischemica-reperfusion injuryin a subject that is about to undergo, is undergoing, or has undergonean organ transplant, provided that the organ transplant is not a kidneytransplant.

In one embodiment, the method according to this aspect of the inventionfurther comprises inhibiting LEA-2-dependent complement activation in asubject experiencing ischemic reperfusion, comprising administering atherapeutically effective amount of a MASP-2 inhibitory agent and aMASP-1, MASP-3, or MASP-1/3 inhibitory agent to the subject. As detailedabove, the use of a combination of pharmacologic agents thatindividually block LEA-1 and LEA-2, is expected to provide an improvedtherapeutic outcome in treating, preventing, or reducing the severity ofischemia reperfusion injuries as compared to the inhibition of LEA-1alone. This outcome can be achieved for example, by co-administration ofan antibody that has LEA-1-blocking activity together with an antibodythat has LEA-2-blocking activity. In some embodiments, LEA-1- andLEA-2-blocking activities are combined into a single molecular entity,and that such entity with combined LEA-1- and LEA-2-blocking activity.Such an entity may comprise or consist of a bispecific antibody whereone antigen-combining site specifically recognizes MASP-1 and blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Alternatively, such an entity may consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes MASP-3 and thus blocks LEA-1 and the secondantigen-combining site specifically recognizes MASP-2 and blocks LEA-2.Such an entity may optimally consist of a bispecific monoclonal antibodywhere one antigen-combining site specifically recognizes both MASP-1 andMASP-3 and thus blocks LEA-1 while the second antigen-combining sitespecifically recognized MASP-2 and blocks LEA-2.

The MASP 2 inhibitory composition may be administered to a subject inneed thereof by intra arterial, intravenous, intracranial,intramuscular, subcutaneous, or other parenteral administration, andpotentially orally for non peptidergic inhibitors, and most suitably byintra arterial or intravenous administration. Administration of theMASP-2 inhibitory compositions of the present invention suitablycommences immediately after or as soon as possible after an ischemiareperfusion event. In instances where reperfusion occurs in a controlledenvironment (e.g., following an aortic aneurism repair, organ transplantor reattachment of severed or traumatized limbs or digits), the MASP-2inhibitory agent may be administered prior to and/or during and/or afterreperfusion. Administration may be repeated periodically as determinedby a physician for optimal therapeutic effect.

Application of the MASP-3 inhibitory compositions and optional MASP 2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treatment or prevention ofischemia reperfusion injuries. Alternatively, the composition may beadministered at periodic intervals such as daily, biweekly, weekly,every other week, monthly or bimonthly over an extended period of timefor treatment of a subject experiencing ischemic reperfusion.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such in a subject experiencing ischemic reperfusion.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingischemia-reperfusion comprising an effective amount of a high affinitymonoclonal antibody or antigen binding fragment thereof as disclosedherein that binds to human MASP-3 and inhibits alternative pathwaycomplement activation to treat or reduce the risk of tissue injuryassociated with ischemia-reperfusion in the subject.

D. The Role of MASP-3 in Inlammatory and Non-inflammatory Arthritidesand Therapeutic Methods Using MASP-3 Inhibitory Antibodies, Optionallyin Combination with and MASP-2 Inhibitory Agents

Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovialjoints that may also have systemic manifestations. RA affectsapproximately 1% of the world population, with women being two to threetimes more likely to be afflicted. Joint inflammation manifests inswelling, pain, and stiffness. As the disease progresses there may bejoint erosion and destruction, resulting in impaired range of motion anddeformities. Treatment goals in RA include prevention or control ofjoint damage, prevention of loss of joint function and diseaseprogression, relief of symptoms and improvement in quality of life, andachievement of drug-free remission. Pharmacological treatment of RAincludes disease-modifying anti-rheumatic drugs (DMARDs), analgesics,and anti-inflammatory agents (glucocorticoids and non-steroidalanti-inflammatory drugs). DMARDs are the most important treatmentbecause they can induce durable remissions and delay or halt theprogression of joint destruction, which is irreversible. TraditionalDMARDs include small molecules such as methotrexate, sulfasalazine,hydroxychloroquine, gold salts, leflunomide, D-penicillamine,cyclosporine, and azathioprine. If traditional DMARDs are inadequate tocontrol the disease then several biologic agents targeting inflammatorycells or mediators are available treatment options, such as tumornecrosis factor inhibitors (etanercept, infliximab, adalimumab,certolizumab pegol, and golimumab), cytokine antagonists (anakinra andtocilizumab), rituximab, and abatacept.

Although adaptive immunity is clearly central to RA pathogenesis asevidenced by genetic association with T-cell activation genes and thepresence of autoantibodies, innate immune mechanisms have also beenimplicated (McInnes, I. B. and Schett, G. New Engl. J. Med.365:2205-2219, 2011). In human RA, synovial fluid levels of thealternative pathway cleavage fragment Bb were several fold higher thansamples from patients with crystal-induced arthritis or degenerativejoint disease, implicating preferential activation of the alternativepathway in RA patients (Brodeur, J. P., et al., Arthritis Rheum.34:1531-1537, 1991). In the experimental anti-type II collagenantibody-passive transfer model of arthritis, factor B-deficient micehad decreased inflammation and joint damage compared with wild-typemice, whereas C4-deficient mice had similar disease activity aswild-type mice, indicating the requirement for the alternative pathwayand not the classical pathway in this model (Banda, N. K. et al., J.Immunol. 177:1904-1912, 2006). In the same experimental model ofcollagen antibody-induced arthritis (CAIA), mice with only classicalpathway active or only lectin pathway active were not capable ofdeveloping arthritis (Banda, N. K. et al., Clin. Exp. Immunol.159:100-108, 2010). Data from this study suggested that either theclassical or lectin pathways were capable of activating low levels of C3in vitro. However, in the absence of the alternative pathwayamplification loop, the level of joint deposition of C3 was inadequateto produce clinical disease. A key step in the activation of thealternative pathway is conversion of the zymogen of factor D (pro-factorD) to mature factor D, which is mediated by MASP-1 and/or MASP-3(Takahashi, M., et al., J. Exp. Med. 207:29-37, 2010) and/or HTRA1(Stanton et al., Evidence That the HTRA1 Interactome InfluencesSusceptibility to Age-Related Macular Degeneration, presented at TheAssociation for Research in Vision and Ophthalmology 2011 conference onMay 4, 2011). The role of MASP-1/3 was evaluated in murine CAIA and theresults showed that MASP-1/3 deficient mice were protected fromarthritis compared with wild-type mice (Banda, N. K., et al., J.Immunol. 185:5598-5606, 2010). In MASP-1/3-deficient mice, pro-factor Dbut not mature factor D was detected in serum during the evolution ofCAIA, and the addition of human factor D in vitro reconstituted C3activation and C5a generation using sera from these mice. In contrast,in a murine model of the effector phase of arthritis, C3-deficient micedeveloped very mild arthritis compared to WT mice while factorB-deficient mice still developed arthritis, indicating independentcontribution of both the classical/lectin and alternative pathways(Hietala, M. A. et al., Eur. J. Immunol. 34:1208-1216, 2004). In theK/BxN T cell receptor transgenic mouse model of inflammatory arthritis,mice lacking C4 or C1q developed arthritis similar to wild-type micewhereas mice lacking factor B either did not develop arthritis or hadmild arthritis, demonstrating the requirement for the alternativepathway and not the classical pathway in this model (Ji H. et al.,Immunity 16:157-168, 2002). In the K/BxN model, mice lacking MBL-A werenot protected from serum-induced arthritis, but as the role of MBL-C wasnot investigated, a potential role for the lectin pathway could not beeliminated (Ji et al., 2002, supra).

Two research groups have independently proposed that lectin-dependentcomplement activation promotes inflammation in RA patients viainteraction of MBL with specific IgG glycoforms (Malhotra et al., Nat.Med. 1:237 243, 1995; Cuchacovich et al., J. Rheumatol. 23:44 51, 1996).It is noted that rheumatoid conditions are associated with a markedincrease in IgG glycoforms that lack galactose (referred to as IgGOglycoforms) in the Fc region of the molecule (Rudd et al., TrendsBiotechnology 22:524 30, 2004). The percentage of IgG0 glycoformsincreases with disease progression of rheumatoid conditions, and returnsto normal when patients go into remission. In vivo, IgG0 is deposited onsynovial tissue and MBL is present at increased levels in synovial fluidin individuals with RA. Aggregated agalactosyl IgG (IgG0) associatedwith RA can bind MBL and therefore can initiate lectin-dependentcomplement activation via LEA-1 and/or LEA-2. Furthermore, results froma clinical study looking at allelic variants of MBL in RA patientssuggest that MBL may have an inflammatory enhancing role in the disease(Garred et al., J. Rheumatol. 27:26 34, 2000). Therefore, thelectin-dependent complement activation via LEA-1 and/or LEA-2 may playan important role in the pathogenesis of RA.

Complement activation also plays in important role in juvenilerheumatoid arthritis (Mollnes, T. E., et al., Arthritis Rheum. 29:135964, 1986). Similar to adult RA, in juvenile rheumatoid arthritis,elevated serum and synovial fluid levels of alternative pathwaycomplement activation product Bb compared to C4d (a marker for classicalor LEA-2 activation), indicate that complement activation is mediatedpredominantly by LEA-1 (El Ghobarey, A. F. et al., J. Rheumatology 7:453460, 1980; Agarwal, A., et al., Rheumatology 39:189 192, 2000).

Similarly, complement activation plays an important role in psoriaticarthritis. Patients with this condition have increased complementactivation products in their circulation, and their red blood cellsappear to have lower levels of the complement regulator CD59 (Triolo,.Clin Exp Rheumatol., 21(2):225-8, 2003). Complement levels areassociated with disease activity, and have a high predictive value todetermine treatment outcomes (Chimenti at al., Clin Exp Rheumatol.,30(1):23-30, 2012). In fact, recent studies suggest that the effect ofanti-TNF therapy for this condition is attributable to complementmodulation (Ballanti et al., Autoimmun Rev., 10(10):617-23, 2011). Whilethe precise role of complement in psoriatic arthritis has not beendetermined, the presence of C4d and Bb complement activation products inthe circulation of these patients suggests an important role inpathogenesis. On the basis of the products observed, it is believed thatLEA-1, and possibly also LEA-2 are responsible for pathologic complementactivation in these patients.

Osteoarthritis (OA) is the most common form of arthritis, affecting over25 million people in the United States. OA is characterized by breakdownand eventual loss of joint cartilage, accompanied by new bone formationand synovial proliferation, leading to pain, stiffness, loss of jointfunction, and disability. Joints that are frequently affected by OA arehands, neck, lower back, knees and hips. The disease is progressive andcurrent treatments are for symptomatic pain relief and do not alter thenatural history of disease. The pathogenesis of OA is unclear, but arole for complement has been implicated. In a proteomic andtranscriptomic analyses of synovial fluid from patients with OA, severalcomponents of complement were aberrantly expressed compared to samplesfrom healthy individuals, including classical (C1s and C4A) andalternative (factor B) pathways, and also C3, CS, C7, and C9 (Wang, Q.,et al., Nat. Med. 17:1674-1679, 2011). Moreover, in a mouse model of OAinduced by medial meniscectomy, CS-deficient mice had less cartilageloss, osteophyte formation and synovitis than CS-positive mice, andtreatment of wild-type mice with CR2-fH, a fusion protein that inhibitsthe alternative pathway, attenuated the development of OA (Wang et al.,2011 supra).

Ross River virus (RRV) and chikungunya virus (CHIKV) belong to a groupof mosquito-borne viruses that can cause acute and persistent arthritisand myositis in humans. In addition to causing endemic disease, theseviruses can cause epidemics that involve millions of infectedindividuals. The arthritis is believed to be initiated by viralreplication and induction of host inflammatory response in the joint andthe complement system has been invoked as a key component in thisprocess. Synovial fluid from humans with RRV-induced polyarthritiscontains higher levels of C3a than synovial fluid from humans with OA(Morrison, T. E., et al., J. Virol. 81:5132-5143, 2007). In a mousemodel of RRV infection, C3-deficient mice developed less severearthritis compared with wild-type mice, implicating the role ofcomplement (Morrison et al., 2007, supra). The specific complementpathway involved was investigated and mice with inactivated lectinpathway (MBL-A−/− and MBL-C−/−) had attenuated arthritis compared withwide-type mice. In contrast, mice with inactivated classical pathway(C1q−/−) or alternative pathway (factor B−/−) developed severearthritis, indicating that the lectin pathway initiated by MBL had anessential role in this model (Gunn, B. M., et al., PLoS Pathog.8:e1002586, 2012). Because arthritides involve damage to the joints, theinitial joint damage caused by various etiologies may trigger asecondary wave of complement activation via LEA-2. In support of thisconcept, our previous work has demonstrated that MASP-2 KO mice havereduced joint injury compared to WT mice in the collagen-induced modelof RA.

In view of the body of evidence detailed above, LEA-1 and LEA-2inhibitors, alone or in combination, are expected to be therapeuticallyuseful for the treatment of arthritides. An optimally effectivetreatment for arthritides may therefore comprise active pharmaceuticalingredients that, alone or in combination, can block both LEA-1 andLEA-2. Combined LEA-1 and LEA-2 inhibition may be accomplished byco-administration of an LEA-1 blocking agent and a LEA2 blocking agent.Preferentially, LEA-1 and LEA-2 inhibitory function may be encompassedin a single molecular entity, such as a bispecific antibody composed ofMASP-1/3 and a MASP-2-specific binding site, or a dual specificityantibody where each binding site can bind to and block MASP-1/3 orMASP-2.In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of inflammatory ornon-inflammatory arthritides, including osteoarthritis, rheumatoidarthritis, juvenile rheumatoid arthritis and psoriatic arthritis, byadministering a composition comprising a therapeutically effectiveamount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent,a MASP-3 inhibitory agent, or a combination of a MASP 1/3 inhibitoryagent, in a pharmaceutical carrier to a subject suffering from, or atrisk for developing, inflammatory or non-inflammatory arthritides. TheMASP-1, MASP-3, or MASP 1/3 inhibitory composition may be administeredto the subject systemically, such as by intra arterial, intravenous,intramuscular, subcutaneous, or other parenteral administration, or byoral administration. Alternatively, administration may be by localdelivery, such as by intra-articular injection. The LEA-1 inhibitoryagent may be administered periodically over an extended period of timefor treatment or control of a chronic condition, or may be by single orrepeated administration in the period before, during and/or followingacute trauma or injury, including surgical procedures performed on thejoint.

In one embodiment, the method according to this aspect of the inventionfurther comprises inhibiting LEA-2-dependent complement activation in asubject suffering from, or at risk for developing, inflammatory ornon-inflammatory arthritides (including osteoarthritis, rheumatoidarthritis, juvenile rheumatoid arthritis and psoriatic arthritis), byadministering a therapeutically effective amount of a MASP-2 inhibitoryagent and a MASP-1, MASP-3, or MASP1/3 inhibitory agent to the subject.As detailed above, the use of a combination of pharmacologic agents thatindividually block LEA-1 and LEA-2, is expected to provide an improvedtherapeutic outcome in treating or preventing arthritides as compared tothe inhibition of LEA-1 alone. This outcome can be achieved for example,by co-administration of an antibody that has LEA-1-blocking activitytogether with an antibody that has LEA-2-blocking activity. In someembodiments, LEA-1- and LEA-2-blocking activities are combined into asingle molecular entity, and that such entity with combined LEA-1- andLEA-2-blocking activity. Such an entity may comprise or consist of abispecific antibody where one antigen-combining site specificallyrecognizes MASP-1 and blocks LEA-1 and the second antigen-combining sitespecifically recognizes MASP-2 and blocks LEA-2. Alternatively, such anentity may consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes MASP-3 and thus blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Such an entity may optimally consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1while the second antigen-combining site specifically recognized MASP-2and blocks LEA-2.

The MASP-2 inhibitory composition may be administered to the subject inneed thereof systemically, such as by intra arterial, intravenous,intramuscular, subcutaneous, or other parenteral administration, orpotentially by oral administration for non peptidergic inhibitors.Alternatively, administration may be by local delivery, such as byintra-articular injection. The MASP-2 inhibitory agent may beadministered periodically over an extended period of time for treatmentor control of a chronic condition, or may be by single or repeatedadministration in the period before, during and/or following acutetrauma or injury, including surgical procedures performed on the joint.

Application of the MASP-3 inhibitory compositions and optional MASP 2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and MASP-3 inhibitory agents, or bispecific ordual-inhibitory agents, or co-administration of separate compositions),or a limited sequence of administrations, for treating, preventing orreducing the severity of inflammatory or non-inflammatory arthritides.Alternatively, the composition may be administered at periodic intervalssuch as daily, biweekly, weekly, every other week, monthly or bimonthlyover an extended period of time for treatment of a subject sufferingfrom inflammatory or non-inflammatory arthritides.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as arthritis.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingarthritis (inflammatory and non-inflammatory arthritides) comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of a high affinity monoclonal antibody or antigenbinding fragment thereof as disclosed herein that binds to human MASP-3and inhibits alternative pathway complement activation to treat orreduce the risk of arthritis in the subject, such as, for example,wherein said antibody or antigen binding fragment thereof comprises (a)a heavy chain variable region comprising (i) VHCDR1 comprising SEQ IDNO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii)VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable regioncomprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ IDNO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO:144 and (iii)VLCDR3 comprising SEQ ID NO:161. In some embodidments, the subject issuffering from arthritis selected fronm the group consisting ofosteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis,ankylosing spondylitis, Behcet's disease, infection-related arthritisand psoriatic arthritis. In some embodiments, the pharmaceuticalcomposition is administered systemically (i.e., subcutaneously,intra-muscularly, intravenously, intra-arterially or as an inhalant). Insome embodiments, the pharmaceutical composition is administered locallyto a joint.

E. The Role of MASPO-3 in Disseminated Intravascular Coagulation (DIC)and Therapeutic Methods Using MASP-3 Inhibitory Antibodies, Optionallyin Combination with and MASP-2 Inhibitory Agents

Disseminated intravascular coagulation (DIC) is a syndrome of pathologicoverstimulation of the coagulation system that can manifest clinicallyas hemorrhage and/or thrombosis. DIC does not occur as a primarycondition but rather in association with a variety of disease processes,including tissue damage (trauma, burns, heat stroke, transfusionreaction, acute transplant rejection), neoplasia, infections, obstetricconditions (placenta previa, amniotic fluid embolism, toxemia ofpregnancy), and miscellaneous conditions such as cardiogenic shock, neardrowning, fat embolism, aortic aneurysm. Thrombocytopenia is a frequentabnormality in patients in the intensive care unit, with an incidence of35% to 44%, and DIC is the etiology in about 25% of these cases, i.e.,DIC occurs in approximately 10% of critically ill patients (Levi, M. andOpal, S. M. Crit. Care 10:222-231, 2006). The pathophysiology of DIC isthat the underlying disease process initiates a physiologicalcoagulation response. However, the prothrombotic substances overwhelmthe normal counterbalancing mechanisms such that there is theinappropriate deposition of fibrin and platelets in themicrocirculation, leading to organ ischemia, hypofibrinogenemia, andthrombocytopenia. The diagnosis of DIC is based on the clinicalpresentation in the appropriate underlying illness or process, alongwith abnormalities in laboratory parameters (prothrombin time, partialthromboplastin time, fibrin degradation products, D-dimer, or plateletcount). The primary treatment of DIC is to address the underlyingcondition that is the responsible trigger. Blood product support in theform of red blood cells, platelets, fresh frozen plasma, andcryoprecipitate may be necessary to treat or prevent clinicalcomplications.

The role of the complement pathways in DIC has been investigated inseveral studies. Complement activation was evaluated in pediatricpatients with meningococcal infection comparing the clinical course inrelation to MBL genotype (Sprong, T. et al., Clin. Infect. Dis.49:1380-1386, 2009). At admission to the hospital, patients with MBLdeficiency had lower circulating levels of C3bc, terminal complementcomplex, C4bc, and C3bBbP than MBL-sufficient patients, indicating lowerextent of common complement, terminal complement, and alternativepathway activation. Furthermore, extent of systemic complementactivation correlated with disease severity and parameters of DIC andthe MBL-deficient patients had a milder clinical course thanMBL-sufficient patients. Therefore, although MBL deficiency is a riskfactor for susceptibility to infections, MBL deficiency during septicshock may be associated with lower disease severity.

As demonstrated in Examples 1-4 herein, experimental studies havehighlighted the important contribution of MBL and MASP-1/3 in innateimmune response to Neisseria menigitidis, the etiological agent ofmeningococcal infection. MBL-deficient sera from mice or humans, MASP-3deficient human sera, or the MASP-1/3 knockout mouse are less effectiveat activating complement and lysing meningococci in vitro compared towild-type sera. Similarly, naïve MASP-1/3 knockout mice are moresusceptible to neisserial infection than their wild-type counterparts.Thus, in the absence of adaptive immunity, the LEA-1 pathway contributesto innate-host resistance to neisserial infection. Conversely, LEA-1augments pathologic complement activation triggering a harmful hostresponse, including DIC.

In a murine model of arterial thrombosis, MBL-null and MASP-1/-3knockout mice had decreased FeC13-induced thrombogenesis compared withwild-type or C2/factor B-null mice, and the defect was reconstitutedwith recombinant human MBL (La Bonte, L. R., et al., J. Immunol.188:885-891, 2012). In vitro, MBL-null or MASP-1/-3 knockout mouse serahad decreased thrombin substrate cleavage compared with wild-type orC2/factor B-null mouse sera; addition of recombinant human MASP-1restored thrombin substrate cleavage in MASP-1/-3 knockout mouse sera(La Bonte et al., 2012, supra). These results indicate that MBL/MASPcomplexes, in particular MASP-1, play a key role in thrombus formation.Thus, LEA-1 may play an important role in pathologic thrombosis,including DIC.

Experimental studies have established an equally important role forLEA-2 in pathologic thrombosis. In vitro studies further demonstratethat LEA-2 provides a molecular link between the complement system andthe coagulation system. MASP-2 has factor Xa-like activity and activatesprothrombin through cleavage to form thrombin, which can subsequentlyclear fibrinogen and promote fibrin clot formation (see also Krarup etal., PLoS One, 18:2(7):e623, 2007).

Separate studies have shown that lectin-MASP complexes can promote clotformation, fibrin deposition and fibrinopeptide release in a MASP-2dependent process (Gulla et al., Immunology, 129(4):482-95, 2010). Thus,LEA-2 promotes simultaneous lectin-dependent activation of complementand the coagulation system.

In vitro studies have further shown that MASP-1 has thrombin-likeactivity (Presanis J. S., et al., Mol. Immunol, 40(13):921-9, 2004), andcleaves fibrinogen and factor XIII (Gulla K. C. et la., Immunology,129(4):482-95, 2010), suggesting that LEA-1 may activate coagulationpathways independently or in concert with LEA-2.

The data detailed above suggest that LEA-1 and LEA-2 provide independentlinks between lectin-dependent complement activation and coagulation.Thus, in view of the above, LEA-1 and LEA-2 inhibitors are expected tohave independent therapeutic benefits in treating a subject sufferingfrom disseminated intravascular coagulation. In some embodiments, thesubject is suffering from disseminated intravascular coagulationsecondary to sepsis, trauma, infection (bacterial, viral, fungal,parasitic), malignancy, transplant rejection, transfusion reaction,obstetric complication, vascular aneurysm, hepatic failure, heat stroke,burn, radiation exposure, shock, or severe toxic reaction (e.g., snakebite, insect bite, transfusion reaction). In some embodiments, thetrauma is a neurological trauma. In some embodiments, the infection is abacterial infection, such as a Neisseria meningitidis infection.

In addition, LEA-1 and LEA-2 inhibitors used together may achieveadditional treatment benefits compared to either agent alone. As bothLEA-1 and LEA-2 are known to be activated by conditions that lead to DIC(for example infection or trauma), LEA-1- and LEA-2-blocking agents,either separately or in combination, are expected to have therapeuticutility in the treatment of DIC. LEA-1 and LEA-2 blocking agents mayprevent different cross-talk mechanisms between complement andcoagulation. LEA-1- and LEA-2-blocking agents may thus havecomplementary, additive or synergistic effects in preventing DIC andother thrombotic disorders.

In addition, LEA-1 and LEA-2 inhibitors used together may achieveadditional treatment benefit compared to either agent alone, or mayprovide effective treatment for a wider spectrum of patient subsets.Combined LEA-1 and LEA-2 inhibition may be accomplished byco-administration of a LEA-1-blocking agent and a LEA-2-blocking agent.Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in asingle molecular entity, such as a bispecific antibody composed ofMASP-1/3 and a MASP-2-specific binding site, or a dual specificityantibody where each binding site and bind to and block MASP-1/3 orMASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of disseminatedintravascular coagulation in a subject in need thereof comprisingadministering a composition comprising a therapeutically effectiveamount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent,a MASP 3 inhibitory agent, or a combination of a MASP-1/3 inhibitoryagent, in a pharmaceutical carrier to a subject experiencing, or at riskfor developing, disseminated intravascular coagulation. The MASP-1,MASP-3, or MASP-1/3 inhibitory composition may be administered to thesubject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled. Fortreatment or prevention of DIC secondary to trauma or other acute event,the LEA-1 inhibitory composition may be administered immediatelyfollowing the traumatic injury or prophylactically prior to, during,immediately following, or within one to seven days or longer, such aswithin 24 hours to 72 hours, after trauma-inducing injury or situationssuch as surgery in patients deemed at risk of DIC. In some embodiments,the LEA-1 inhibitory composition may suitably be administered in a fastacting dosage form, such as by intravenous or intra arterial delivery ofa bolus of a solution containing the LEA-1 inhibitory agent composition.

In one embodiment, the method according to this aspect of the inventionfurther comprises inhibiting LEA-2-dependent complement activation fortreating, preventing, or reducing the severity of disseminatedintravascular coagulation in a subject in need thereof, comprisingadministering a therapeutically effective amount of a MASP-2 inhibitoryagent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to the subject.As detailed above, the use of a combination of pharmacologic agents thatindividually block LEA-1 and LEA-2 is expected to provide an improvedtherapeutic outcome in treating or preventing disseminated intravascularcoagulation as compared to the inhibition of LEA-1 alone. This outcomecan be achieved for example, by co-administration of an antibody thathas LEA-1-blocking activity together with an antibody that hasLEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blockingactivities are combined into a single molecular entity, and that suchentity with combined LEA-1- and LEA-2-blocking activity. Such an entitymay comprise or consist of a bispecific antibody where oneantigen-combining site specifically recognizes MASP-1 and blocks LEA-1and the second antigen-combining site specifically recognizes MASP-2 andblocks LEA-2. Alternatively, such an entity may consist of a bispecificmonoclonal antibody where one antigen-combining site specificallyrecognizes MASP-3 and thus blocks LEA-1 and the second antigen-combiningsite specifically recognizes MASP-2 and blocks LEA-2. Such an entity mayoptimally consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes both MASP-1 and MASP-3and thus blocks LEA-1 while the second antigen-combining sitespecifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subject in needthereof systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled. ForDIC secondary to trauma or other acute event, the MASP-2 inhibitorycomposition may be administered immediately following the traumaticinjury or prophylactically prior to, during, immediately following, orwithin one to seven days or longer, such as within 24 hours to 72 hours,after trauma-inducing injury or situations such as surgery in patientsdeemed at risk of DIC. In some embodiments, the MASP-2 inhibitorycomposition may suitably be administered in a fast acting dosage form,such as by intravenous or intra arterial delivery of a bolus of asolution containing the MASP-2 inhibitory agent composition.

Application of the MASP-3 inhibitory compositions and optional MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and MASP-3 inhibitory agents, or bispecific ordual-inhibitory agents, or co-administration of separate compositions),or a limited sequence of administrations, for treating, preventing, orreducing the severity of disseminated intravascular coagulation insubject in need thereof Alternatively, the composition may beadministered at periodic intervals such as daily, biweekly, weekly,every other week, monthly or bimonthly over an extended period of timefor treatment of a subject experiencing, or at risk for developingdisseminated intravascular coagulation.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as disseminated intravascular coagulation.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingdisseminated intravascular coagulation comprising an effective amount ofa high affinity monoclonal antibody or antigen binding fragment thereofas disclosed herein that binds to human MASP-3 and inhibits alternativepathway complement activation to treat or reduce the risk of developingdisseminated intravascular coagulation, such as, for example, whereinsaid antibody or antigen binding fragment thereof comprises (a) a heavychain variable region comprising (i) VHCDR1 comprising SEQ ID NO:84,(ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3comprising SEQ ID NO:88; and (b) a light chain variable regioncomprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ IDNO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO:144 and (iii)VLCDR3 comprising SEQ ID NO:161.

F. The Role of MASP-3 in Thrombotic Microangiopathy (TMA), IncludingHemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome(AHUS) and Thrombotic Thrombocytopenic Purpura (TTP) and TherapeuticMethods Using MASP-3 Inhibitory Antibodies, Optionally in Combinationwith MASP-2 Inhibitory Agents

Thrombotic microangiopathy (TMA) refers to a group of disorderscharacterized clinically by thrombocytopenia, microangiopathic hemolyticanemia, and variable organ ischemia. The characteristic pathologicalfeatures of TMA are platelet activation and the formation ofmicrothrombi in the small arterioles and venules. The classic TMAs arehemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura(TTP). HUS is distinguished from TTP by the presence of acute renalfailure. HUS occurs in two forms: diarrhea-associated (D+) or typicalHUS, and diarrhea negative (D−) or atypical HUS (aHUS).

HUS

D+HUS is associated with a prodromal diarrheal illness usually caused byEscherichia coli O157 or another Shiga-toxin-producing strain ofbacteria, accounts for over 90% of the HUS cases in children, and is themost common cause of acute renal failure in children. Although humaninfection with Escherichia coli O157 is relatively frequent, thepercentages of bloody diarrhea that progresses to D+HUS ranged from 3%to 7% in sporadic cases and 20% to 30% in some outbreaks (Zheng, X. L.and Sadler, J. E., Annu. Rev. Pathol. 3:249-277, 2008). HUS usuallyoccurs 4 to 6 days after the onset of diarrhea and approximatelytwo-third of children require dialysis in the acute phase of thedisease. Treatment of D+HUS is supportive as no specific treatments havebeen shown to be effective. The prognosis of D+HUS is favorable, withthe majority of patients regaining renal function.

The pathogenesis of D+HUS involves bacteria-produced Shiga toxins thatbind to membranes on microvascular endothelial cells, monocytes, andplatelets. The microvasculature of the kidney is most often affected.Following binding, the toxin is internalized, leading to release ofproinflammatory mediators and eventual cell death. It is thought thatendothelial cell damage triggers renal microvascular thrombosis bypromoting the activation of the coagulation cascade. There is evidencefor activation of the complement system in D+HUS. In children withD+HUS, plasma levels of Bb and SC5b-9 were increased at the time ofhospitalization compared to normal controls and, at day 28 afterhospital discharge, the plasma levels had normalized (Thurman, J. M. etal., Clin. J. Am. Soc. Nephrol. 4:1920-1924, 2009). Shiga toxin 2 (Stx2)was found to activate human complement in the fluid phase in vitro,predominantly via the alternative pathway as activation proceeded in thepresence of ethylene glycol tetraacetic acid which blocks the classicalpathway (Orth, D. et al., J. Immunol. 182:6394-6400, 2009). Furthermore,Stx2 bound factor H and not factor I, and delayed the cofactor activityof factor H on cell surfaces (Orth et al, 2009, supra). These resultssuggest that Shiga toxin may cause renal damage through multiplepotential mechanisms, including a direct toxic effect, and indirectlythrough activation of complement or inhibition of complement regulators.Toxic effects on the vascular endothelium are expected to activatecomplement via LEA-2, as evidenced by the effectiveness of MASP-2blockade in preventing complement-mediated reperfusion injury in variousvascular beds as described in Schwaeble, W. J., et al., Proc. Natl.Acad. Sci. 108:7523-7528, 2011.

In a murine model of HUS induced by co-injection of Shiga toxin andlipopolysaccharide, factor B-deficient mice had less thrombocytopeniaand were protected from renal impairment compared with wild-type mice,implicating LEA-1-dependent activation of the alternative pathway inmicrovascular thrombosis (Morigi, M. et al., J. Immunol. 187:172-180,2011). As described herein, in the same model, administration of MASP-2antibody was also effective and increased survival following STXchallenge, implicating LEA-2-dependent complement pathway inmicrovascular thrombosis.

Based on the foregoing, LEA-1 and LEA-2 inhibitors are expected to haveindependent therapeutic benefit in the treatment or prevention of HUS.In addition, LEA-1 and LEA-2 inhibitors used together may achieveadditional treatment benefit compared to either agent alone, or mayprovide effective treatment for a wider spectrum of patient subsets.Combined LEA-1 and LEA-2 inhibition may be accomplished byco-administration of a LEA-1-blocking agent and a LEA-2-blocking agent.Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in asingle molecular entity, such as a bispecific antibody composed ofMASP-1/3 and a MASP-2-specific binding site, or a dual-specificityantibody where each binding site can bind to and block MASP-1/3 orMASP-2.

aHUS

Atypical HUS is a rare disease, with an estimated incidence of 2 permillion in the United States (Loirat, C. and Fremeaux-Bacchi, V.Orphaned. J. Rare Dis. 6:60-90, 2011). Atypical HUS can develop at anyage, although the majority of patients have an onset during childhood.Atypical HUS is heterogeneous: some cases are familial, some arerecurring, and some are triggered by an infectious illness, typicallyupper respiratory tract or gastroenteritis. The onset of aHUS is usuallysudden and most patients require dialysis at admission. Extra renalmanifestations are present in about 20% of patients and may involve thecentral nervous system, myocardial infarction, distal ischemic gangrene,or multiorgan failure. Treatment of aHUS includes supportive care fororgan dysfunction, plasma infusion or plasma exchange, and eculizumab, ahumanized monoclonal antibody that targets C5 that was recently approvedfor use in the United States and European Union. The prognosis in aHUSis not as good as in D+HUS, with approximately 25% mortality during theacute stage and most survivors develop end-stage renal disease.

Atypical HUS has been characterized as a disease of complementdysregulation in that approximately 50% of patients have mutations ingenes encoding complement regulatory proteins (Zheng and Sadler, 2008supra). Most mutations are seen in factor H (FH); other mutationsinclude membrane cofactor protein (MCP), factor I (FI), factor B, andC3. Functional studies showed that the mutations in FH, MCP, and FI leadto loss of function and therefore more complement activation, whereasmutations in factor B are gain of function. The effects of thesemutations predominantly affect the alternative pathway. These geneticabnormalities are risk factors rather than the only cause of disease asapproximately 50% of family members who carry the mutation do notpresent with the disease by age 45 (Loirat and Fremeaux-Bacchi, 2011supra).

Factor H is a complement control protein that protects host tissue fromalternative pathway complement attack. FH regulates the alternativepathway amplification loop in three ways: it is a cofactor for FI, whichcleaves C3b, it inhibits the formation of the alternative pathway C3convertase, C3bBb, and it binds to polyanions on cell surfaces andtissue matrices and blocks deposition of C3b (Atkinson, J. P. andGoodship, T. H. J., J. Exp. Med. 6:1245-1248, 2007). The majority of FHmutations in aHUS patients occur in the C-terminal short consensusrepeat domains of the protein, which result in defective binding of FHto heparin, C3b, and endothelium, but do not alter plasma C3 regulationwhich resides among N-terminal domains (Pickering, M. C. et al., J. Exp.Med. 204:1249-1256, 2007). FH-deficient mice have uncontrolled plasma C3activation and spontaneously develop membranoproliferativeglomerulonephritis type II, but not aHUS. However, FH-deficient micethat transgenically expressed a mouse FH protein functionally equivalentto aHUS-associated human FH mutants spontaneously develop a HUS but notmembranoproliferative glomerulonephritis type II, providing in vivoevidence that defective control of alternative pathway activation inrenal endothelium is a key event in the pathogenesis of FH-associatedaHUS (Pickering et al., 2007 supra). Another form of FH-associated aHUSoccurs in patients who have anti-FH autoantibodies resulting in a lossof FH functional activity; most of these patients have deletions ingenes encoding five FH-related proteins (Loirat and Fremeaux-Bacchi,2011, supra).

Similar to FH, MCP inhibits complement activation by regulating C3bdeposition on target cells. MCP mutations result in proteins with lowC3b-binding and cofactor activity, thus allowing for dysregulatedalternative pathway activation. FI is a serine protease that cleaves C3band C4b in the presence of cofactors, such as FH and MCP, and therebyprevents the formation of C3 and C5 convertases and inhibits both thealternative and the classical complement pathways. Most of theFI-associated aHUS mutations result in reduced FI activity for thedegradation of C3b and C4b (Zheng and Stadler, 2008, supra). FB is azymogen that carries the catalytic sites of the alternative pathwayconvertase C3bBb. Functional analysis showed that the aHUS associated FBmutations result in increased alternative pathway activation (Loirat andFremeaux-Bacchi, 2011, supra). Heterozygous mutations in C3 areassociated with aHUS. Most C3 mutations induce a defect of C3 to bindMCP, leading to an increased capacity of FB to bind C3b and increasedformation of C3 convertase (Loirat and Fremeaux-Bacchi, 2011, supra).Thus, aHUS is a disease closely associated with mutations in thecomplement genes that lead to inadequate control of the alternativepathway amplification loop. Since the alternative pathway amplificationloop is dependent on factor B proteolytic activity, and since LEA-1 isrequired for factor B activation (either by MASP-3 dependent cleavage orby factor D-mediated cleavage wherein the MASP-1 contributes to thematuration of factor D), LEA-1-blocking agents are expected to preventuncontrolled complement activation in susceptible individuals. As aresult, it is expected that LEA-1 blocking agents will effectively treataHUS.

While the central role of a deregulated alternative pathwayamplification loop in aHUS is widely accepted, the triggers initiatingcomplement activation and the molecular pathways involved areunresolved. Not all individuals carrying the above-described mutationsdevelop aHUS. In fact, familial studies have suggested that thepenetrance of aHUS is only ˜50% (Sullivan M. et al., Ann Hum Genet74:17-26 2010). The natural history of the disease suggests that aHUSmost often develops after an initiating event such as an infectiousepisode or an injury. Infectious agents are well known to activate thecomplement system. In the absence of pre-existing adaptive immunity,complement activation by infectious agents may be primarily initiatedvia LEA-1 or LEA-2. Thus, lectin-dependent complement activationtriggered by an infection may represent the initiating trigger forsubsequent pathological amplification of complement activation inaHUS-predisposed individuals, which may ultimately lead to diseaseprogression. Accordingly, another aspect of the present inventioncomprises treating a patient suffering with aHUS secondary to aninfection by administering an effective amount of a LEA-1- or aLEA-2-inhibitory agent.

Other forms of injury to host tissue will activate complement via LEA-2,in particular injury to the vascular endothelium. Human vascularendothelial cells subject to oxidative stress, for example, respond byexpressing surface moieties that bind lectins and activate the LEA-2pathway of complement (Collard et al., Am J Pathol 156(5):1549-56,2000). Vascular injury following ischemia/reperfusion also activatescomplement via LEA-2 in vivo (Moller-Kristensen et al., Scand J Immunol61(5):426-34, 2005). Lectin pathway activation in this setting haspathological consequences for the host, and as shown in Examples 22 and23, inhibition of LEA-2 by blocking MASP-2 prevents further host tissueinjury and adverse outcomes (see also Schwaeble PNAS, 2011, supra).

Thus, other processes that precipitate aHUS are also known to activateLEA-1 or LEA-2. It is therefore likely that the LEA-1 and/or LEA-2pathway may represent the initial complement activating mechanism thatis inappropriately amplified in a deregulated fashion in individualsgenetically predisposed to aHUS, thus initiating aHUS pathogenesis. Byinference, agents that block activation of complement via LEA-1 and/orLEA-2 are expected to prevent disease progression or reduceexacerbations in aHUS susceptible individuals.

In further support of this concept, recent studies have identifiedStreptococcus-pneumoniae as an important etiological agent in pediatriccases of aHUS. (Lee, C. S. et al, Nephrology, 17(1):48-52 (2012);Banerjee R. et al., Pediatr Infect Dis J., 30(9):736-9 (2011)). Thisparticular etiology appears to have an unfavorable prognosis, withsignificant mortality and long-term morbidity. Notably, these casesinvolved non-enteric infections leading to manifestations ofmicroangiopathy, uremia and hemolysis without evidence of concurrentmutations in complement genes known to predispose to aHUS. It isimportant to note that S. pneumoniae is particularly effective atactivating complement, and does so predominantly through LEA-2. Thus, incases of non-enteric HUS associated with pneumococcal infection,manifestations of microangiopathy, uremia and hemolysis are expected tobe driven predominantly by activation of LEA-2, and agents that blockLEA-2, including MASP-2 antibodies, are expected to prevent progressionof aHUS or reduce disease severity in these patients. Accordingly,another aspect of the present invention comprises treating a patientsuffering with non-enteric aHUS that is associated with S. pneumoniaeinfection by administering an effective amount of a MASP-2 inhibitoryagent.

TTP

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorderof the blood-coagulation system caused by autoimmune or hereditarydysfunctions that activate the coagulation cascade or the complementsystem (George, J N, N Engl J Med; 354:1927-35, 2006). This results innumerous microscopic clots, or thomboses, in small blood vesselsthroughout the body, which is a characteristic feature of TMAs. Redblood cells are subjected to shear stress, which damages theirmembranes, leading to intravascular hemolysis. The resulting reducedblood flow and endothelial injury results in organ damage, includingbrain, heart, and kidneys. TTP is clinically characterized bythrombocytopenia, microangiopathic hemolytic anemia, neurologicalchanges, renal failure and fever. In the era before plasma exchange, thefatality rate was 90% during acute episodes. Even with plasma exchange,survival at six months is about 80%.

TTP may arise from genetic or acquired inhibition of the enzymeADAMTS-13, a metalloprotease responsible for cleaving large multimers ofvon Willebrand factor (vWF) into smaller units. ADAMTS-13 inhibition ordeficiency ultimately results in increased coagulation (Tsai, H. J AmSoc Nephrol 14: 1072-1081, 2003). ADAMTS-13 regulates the activity ofvWF; in the absence of ADAMTS-13, vWF forms large multimers that aremore likely to bind platelets and predisposes patients to plateletaggregation and thrombosis in the microvasculature.

Numerous mutations in ADAMTS13 have been identified in individuals withTTP. The disease can also develop due to autoantibodies againstADAMTS-13. In addition, TTP can develop during breast, gastrointestinaltract, or prostate cancer (George J N., Oncology (Williston Park).25:908-14, 2011), pregnancy (second trimester or postpartum), (George JN., Curr Opin Hematol 10:339-344, 2003), or is associated with diseases,such as HIV or autoimmune diseases like systemic lupus erythematosis(Hamasaki K, et al., Clin Rheumatol. 22:355-8, 2003). TTP can also becaused by certain drug therapies, including heparin, quinine, immunemediated ingredient, cancer chemotherapeutic agents (bleomycin,cisplatin, cytosine arabinoside, daunomycin gemcitabine, mitomycin C,and tamoxifen), cyclosporine A, oral contraceptives, penicillin,rifampin and anti-platelet drugs including ticlopidine and clopidogrel(Azarm, T. et al., J Res Med Sci., 16: 353-357, 2011). Other factors orconditions associated with TTP are toxins such as bee venoms, sepsis,splenic sequestration, transplantation, vasculitis, vascular surgery,and infections like Streptococcus pneumoniae and cytomegalovirus (MoakeJ L., N Engl J Med., 347:589-600, 2002). TTP due to transient functionalADAMTS-13 deficiency can occur as a consequence of endothelial cellinjury associated with S. pneumoniae infection (Pediatr Nephrol,26:631-5, 2011).

Plasma exchange is the standard treatment for TTP (Rock G A, et al., NEngl J Med 325:393-397, 1991). Plasma exchange replaces ADAMTS-13activity in patients with genetic defects and removes ADAMTS-13autoantibodies in those patients with acquired autoimmune TTP (Tsai,H-M, Hematol Oncol Clin North Am., 21(4): 609-v, 2007). Additionalagents such as immunosuppressive drugs are routinely added to therapy(George, J N, N Engl J Med, 354:1927-35, 2006). However, plasma exchangeis not successful for about 20% of patients, relapse occurs in more thana third of patients, and plasmapheresis is costly and technicallydemanding. Furthermore, many patients are unable to tolerate plasmaexchange. Consequently, there remains a critical need for additional andbetter treatments for TTP.

Because TTP is a disorder of the blood coagulation cascade, treatmentwith antagonists of the complement system may aid in stabilizing andcorrecting the disease. While pathological activation of the alternativecomplement pathway is linked to aHUS, the role of complement activationin TTP is less clear. The functional deficiency of ADAMTS13 is importantfor the susceptibility to TTP, however it is not sufficient to causeacute episodes. Environmental factors and/or other genetic variationsmay contribute to the manifestation of TTP. For example, genes encodingproteins involved in the regulation of the coagulation cascade, vWF,platelet function, components of the endothelial vessel surface, or thecomplement system may be implicated in the development of acutethrombotic microangiopathy (Galbusera, M. et al., Haematologica, 94:166-170, 2009). In particular, complement activation has been shown toplay a critical role; serum from thrombotic microangiopathy associatedwith ADAMTS-13 deficiency has been shown to cause C3 and MAC depositionand subsequent neutrophil activation which could be abrogated bycomplement inactivation (Ruiz-Torres M P, et al., Thromb Haemost,93:443-52, 2005). In addition, it has recently been shown that duringacute episodes of TTP there are increased levels of C4d, C3bBbP, and C3a(M. Rai et al., J Thromb Haemost. 10(5):791-798, 2012), consistent withactivation of the classical, lectin and alternative pathways. Thisincreased amount of complement activation in acute episodes may initiatethe terminal pathway activation and be responsible for furtherexacerbation of TTP.

The role of ADAMTS-13 and vWF in TTP clearly is responsible foractivation and aggregation of platelets and their subsequent role inshear stress and deposition in microangiopathies. Activated plateletsinteract with and trigger both the classical and alternative pathways ofcomplement. Platelet-mediated complement activation increases theinflammatory mediators C3a and C5a (Peerschke E. et al., Mol Immunol,47:2170-5 (2010)). Platelets may thus serve as targets of classicalcomplement activation in inherited or autoimmune TTP.

As described above, the lectin-dependent activation of complement, byvirtue of the thrombin-like activity of MASP-1 and the LEA-2-mediatedprothombin activation, is the dominant molecular pathway linkingendothelial injury to the coagulation and microvascular thrombosis thatoccurs in HUS. Similarly, activation of LEA-1 and LEA-2 may directlydrive the coagulation system in TTP. LEA-1 and LEA-2 pathway activationmay be initiated in response to the initial endothelium injury caused byADAMTS-13 deficiency in TTP. It is therefore expected that LEA-1 andLEA-2 inhibitors, including but not limited to antibodies that blockMASP-2 function, MASP-1 function, MASP-3 function, or MASP-1 and MASP-3function will mitigate the microangiopathies associated withmicrovascular coagulation, thrombosis, and hemolysis in patientssuffering from TTP.

Patients suffering from TTP typically present in the emergency room withone or more of the following: purpura, renal failure, low platelets,anemia and/or thrombosis, including stroke. The current standard of carefor TTP involves intra-catheter delivery (e.g., intravenous or otherform of catheter) of replacement plasmapheresis for a period of twoweeks or longer, typically three times a week, but up to daily. If thesubject tests positive for the presence of an inhibitor of ADAMTS13(i.e., an endogenous antibody against ADAMTS13), then the plasmapheresismay be carried out in combination with immunosuppressive therapy (e.g.,corticosteroids, rituxan, or cyclosporine). Subjects with refractory TTP(approximately 20% of TTP patients) do not respond to at least two weeksof plasmapheresis therapy.

In accordance with the foregoing, in one embodiment, in the setting ofan initial diagnosis of TTP, or in a subject exhibiting one or moresymptoms consistent with a diagnosis of TTP (e.g., central nervoussystem involvement, severe thrombocytopenia (a platelet count of lessthan or equal to 5000/μL if off aspirin, less than or equal to 20,000/μLif on aspirin), severe cardiac involvement, severe pulmonaryinvolvement, gastro-intestinal infarction or gangrene), a method isprovided for treating the subject with an effective amount of a LEA-2inhibitory agent (e.g., a MASP-2 antibody) or a LEA-1 inhibitory agent(e.g., a MASP-1 or MASP-3 antibody) as a first line therapy in theabsence of plasmapheresis, or in combination with plasmapheresis. As afirst-line therapy, the LEA-1 and/or LEA-2 inhibitory agent may beadministered to the subject systemically, such as by intra arterial,intravenous, intramuscular, inhalational, nasal, subcutaneous or otherparenteral administration. In some embodiments, the LEA-1 and/or LEA-2inhibitory agent is administered to a subject as a first-line therapy inthe absence of plasmapheresis to avoid the potential complications ofplasmapheresis, such as hemorrhage, infection, and exposure to disordersand/or allergies inherent in the plasma donor, or in a subject otherwiseaverse to plasmapheresis, or in a setting where plasmapheresis isunavailable. In some embodiments, the LEA-1 and/or LEA-2 inhibitoryagent is administered to the subject suffering from TTP in combination(including co-administration) with an immunosuppressive agent (e.g.,corticosteroids, rituxan or cyclosporine) and/or in combination withconcentrated ADAMTS-13.

In some embodiments, the method comprises administering a LEA-1 and/orLEA-2 inhibitory agent to a subject suffering from TTP via a catheter(e.g., intravenously) for a first time period (e.g., an acute phaselasting at least one day to a week or two weeks) followed byadministering a LEA-1 and/or LEA-2 inhibitory agent to the subjectsubcutaneously for a second time period (e.g., a chronic phase of atleast two weeks or longer). In some embodiments, the administration inthe first and/or second time period occurs in the absence ofplasmapheresis. In some embodiments, the method is used to maintain thesubject to prevent the subject from suffering one or more symptomsassociated with TTP.

In another embodiment, a method is provided for treating a subjectsuffering from refractory TTP (i.e., a subject that has not responded toat least two weeks of plasmaphoresis therapy), by administering anamount of a LEA-1 and/or LEA-2 inhibitor effective to reduce one or moresymptoms of TTP. In one embodiment, the LEA-1 and/or LEA-2 inhibitor isadministered to a subject with refractory TTP on a chronic basis, over atime period of at least two weeks or longer via subcutaneous or otherparenteral administration. Administration may be repeated as determinedby a physician until the condition has been resolved or is controlled.

In some embodiments, the method further comprises determining the levelof at least one complement factor (e.g., C3, C5) in the subject prior totreatment, and optionally during treatment, wherein the determination ofa reduced level of the at least one complement factor in comparison to astandard value or healthy control subject is indicative of the need forcontinued treatment with the LEA-1 and/or LEA-2 inhibitory agent.

In some embodiments, the method comprises administering, eithersubcutaneously or intravenously, a LEA-1 and/or LEA-2 inhibitory agentto a subject suffering from, or at risk for developing, TTP. Treatmentis preferably daily, but can be as infrequent as monthly. Treatment iscontinued until the subject's platelet count is greater than 150,000/m1for at least two consecutive days.

In summary, LEA-1 and LEA-2 inhibitors are expected to have independenttherapeutic benefit in the treatment of TMAs, including HUS, aHUS andTTP. In addition, LEA-1 and LEA-2 inhibitors used together are expectedto achieve additional treatment benefit compared to either agent alone,or may provide effective treatment for a wider spectrum of patientsubsets suffering from variant forms of TMA. Combined LEA-1 and LEA-2inhibition may be accomplished by co-administration of a LEA-1 blockingagent and a LEA2 blocking agent. Optimally, LEA-1 and LEA-2 inhibitoryfunction may be encompassed in a single molecular entity, such as abispecific antibody composed of MASP-1/3 and a MASP-2-specific bindingsite, or a dual specificity antibody where each binding site can bind toand block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of a thromboticmicroangiopathy, such as hemolytic uremic syndrome (HUS), atypicalhemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura(TTP) comprising administering a composition comprising atherapeutically effective amount of a LEA-1 inhibitory agent comprisinga MASP 1 inhibitory agent, a MASP 3 inhibitory agent, or a combinationof a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subjectsuffering from, or at risk for developing a thrombotic microangiopathy.The MASP 1, MASP 3, or MASP 1/3 inhibitory composition may beadministered to the subject systemically, such as by intra arterial,intravenous, intramuscular, inhalational, nasal, subcutaneous or otherparenteral administration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

In one embodiment, the method according to this aspect of the inventionfurther comprises inhibiting LEA-2-dependent complement activation fortreating, preventing, or reducing the severity of a thromboticmicroangiopathy, such as hemolytic uremic syndrome (HUS), atypicalhemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura(TTP) comprising administering a therapeutically effective amount of aMASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitoryagent to a subject suffering from, or at risk for developing athrombotic microangiopathy. As detailed above, the use of a combinationof pharmacologic agents that individually block LEA-1 and LEA-2, isexpected to provide an improved therapeutic outcome in treating orpreventing or reducing the severity of a thrombotic microangiopathy ascompared to the inhibition of LEA-1 alone. This outcome can be achievedfor example, by co-administration of an antibody that has LEA-1-blockingactivity together with an antibody that has LEA-2-blocking activity. Insome embodiments, LEA-1- and LEA-2-blocking activities are combined intoa single molecular entity, and that such entity with combined LEA-1- andLEA-2-blocking activity. Such an entity may comprise or consist of abispecific antibody where one antigen-combining site specificallyrecognizes MASP-1 and blocks LEA-1 and the second antigen-combining sitespecifically recognizes MASP-2 and blocks LEA-2. Alternatively, such anentity may consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes MASP-3 and thus blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Such an entity may optimally consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1while the second antigen-combining site specifically recognized MASP-2and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subjectsystemically, such as by intra arterial, intravenous, intramuscular,inhalational, nasal, subcutaneous or other parenteral administration, orpotentially by oral administration for non peptidergic agents.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and optional MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treating, preventing orreducing the severity of a thrombotic microangiopathy in a subjectsuffering from, or at risk for developing, a thrombotic microangiopathy.Alternatively, the composition may be administered at periodic intervalssuch as daily, biweekly, weekly, every other week, monthly or bimonthlyover an extended period of time for treatment of a subject in needthereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as a thrombotic microangiopathy (e.g., hemolytic uremicsyndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or thromboticthrombocytopenic purpura (TTP).

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developing athrombotic microangiopathy (e.g.,hemolytic uremic syndrome (HUS),atypical hemolytic uremic syndrome (aHUS), or thromboticthrombocytopenic purpura (TTP), comprising an effective amount of a highaffinity monoclonal antibody or antigen binding fragment thereof asdisclosed herein that binds to human MASP-3 and inhibits alternativepathway complement activation to treat or reduce the risk of developinga thrombotic microangiopathy (e.g., hemolytic uremic syndrome (HUS),atypical hemolytic uremic syndrome (aHUS), thrombotic thrombocytopenicpurpura (TTP), or transplant-related TMA (TA-TMA), such as, for example,wherein said antibody or antigen binding fragment thereof comprises (a)a heavy chain variable region comprising (i) VHCDR1 comprising SEQ IDNO:84, (ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii)VHCDR3 comprising SEQ ID NO:88; and (b) a light chain variable regioncomprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ IDNO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO:144 and (iii)VLCDR3 comprising SEQ ID NO:161.

G. The Role of Masp-3 in Asthma and Therapeutic Methods Using MASP-3Inhibitory Antibodies, Optionally in Combination with MASP-2 InhibitoryAgents

Asthma is a common chronic inflammatory disease of the airways.Approximately 25 million people in the United States have asthma,including seven million children under the age of 18, with more thanhalf experiencing at least one asthma attack each year, leading to morethan 1.7 million emergency department visits and 450,000hospitalizations annually (world-wide-web atgov/health/prof/lung/asthma/naci/asthma-info/index.htm., accessed on May4, 2012). The disease is heterogeneous with multiple clinicalphenotypes. The most common phenotype is allergic asthma. Otherphenotypes include nonallergic asthma, aspirin-exacerbated respiratorydisease, post-infectious asthma, occupational asthma, airborneirritant-induced asthma, and exercise-induced asthma. The cardinalfeatures of allergic asthma include airway hyperresponsiveness (AHR) toa variety of specific and nonspecific stimuli, excessive airway mucusproduction, pulmonary eosinophilia, and elevated concentration of serumIgE. The symptoms of asthma include coughing, wheezing, chest tightness,and shortness of breath. The goal of asthma treatment is to control thedisease and minimize exacerbations, daily symptoms, and allow patientsto be physically active. Current treatment guidelines recommend stepwisetreatments until asthma control is attained. The first treatment step isas needed rapid-acting inhaled β2-agonist, followed by addition ofcontroller medications such as inhaled corticosteroids, long-actinginhaled β2-agonists, leukotriene modifier drugs, theophylline, oralglucocorticosteroids, and anti-IgE monoclonal antibody.

Although asthma is multifactorial in origin, it is generally acceptedthat it arises as a result of inappropriate immunological responses tocommon environmental antigens in genetically susceptible individuals.Asthma is associated with complement activation and the anaphylatoxins(AT) C3a and C5a have proinflammatory and immunoregulatory propertiesthat are relevant to the development and modulation of the allergicresponse (Zhang, X. and Kohl, J. Expert. Rev. Clin. Immunol., 6:269-277,2010). However, the relative involvement of the classical, alternative,and lectin pathways of complement in asthma is not well understood. Thealternative pathway may be activated on the surface of allergens and thelectin pathway may be activated through recognition of allergenpolysaccharide structures, both processes leading to the generation ofAT. Complement may be activated by different pathways depending on thecausative allergen involved. Highly allergic grass pollen of theParietaria family for example is very effective at promotingMBL-dependent activation of C4, implicating LEA-2. Conversely, housedust mite allergen does not require MBL for complement activation (Vargaet al. Mol Immunol., 39(14):839-46, 2003).

Environmental triggers of asthma may activate complement by thealternative pathway. For example, in vitro exposure of human serum tocigarette smoke or diesel exhaust particles resulted in activation ofcomplement and the effect was unaffected by the presence of EDTA,suggesting activation was via the alternative rather than classicalpathway (Robbins, R. A. et al, Am. J. Physiol. 260: L254-L259, 1991;Kanemitsu, H., et al., Biol. Pharm. Bull. 21:129-132, 1998). The role ofcomplement pathways in allergic airway inflammation was evaluated in amouse ovalbumin sensitization and challenge model. Wild-type micedeveloped AHR and airway inflammation in response to aeroallergenchallenge. A Crry-Ig fusion protein which inhibits all pathways ofcomplement activation, was effective in preventing AHR and lunginflammation when administered systemically or locally by inhalation inthe mouse ovalbumine model of allergic lung inflammation (Taube et al.,Am J Respir Crit Care Med., 168(11):1333-41, 2003).

In comparison to wild-type mice, factor B-deficient mice demonstratedless AHR and airway inflammation whereas C4-deficient mice had similareffects as wild-type mice (Taube, C., et al., Proc. Natl. Acad. Sci. USA103:8084-8089, 2006). These results support a role for alternativepathway and not classical pathway involvement in the murine aeroallergenchallenge model. Further evidence for the importance of the alternativepathway was provided in a study of factor H (FH) using the same mousemodel (Takeda, K., et al., J. Immunol. 188:661-667, 2012). FH is anegative regulator of the alternative pathway and acts to preventautologous injury of self tissues. Endogenous FH was found to be presentin airways during allergen challenge and inhibition of FH with arecombinant competitive antagonist increased the extent of AHR andairway inflammation (Takeda et al., 2012, supra). Therapeutic deliveryof CR2-fH, a chimeric protein that links the iC3b/C3d binding region ofCR2 to the complement-regulatory region of FH which targets thecomplement regulatory activity of fH to sites of existing complementactivation, protected the development of AHR and eosinophil infiltrationinto the airways after allergen challenge (Takeda et al., 2012, supra).The protective effect was demonstrated with ovalbumin as well as ragweedallergen, which is a relevant allergen in humans.

The role of lectin-dependent complement activation in asthma wasevaluated in a mouse model of fungal asthma (Hogaboam et al., J.Leukocyte Biol. 75:805 814, 2004). These studies used mice geneticallydeficient in mannan binding lectin A (MBL-A), a carbohydrate bindingprotein that functions as the recognition component for activation ofthe lectin complement pathways. MBL-A(+/+) and MBL-A(−/−) Aspergillus,fumigatus sensitized mice were examined at days 4 and 28 after an i.t.challenge with A. fumigatus conidia. AHR in sensitized MBL-A(−/−) micewas significantly attenuated at both times after conidia challengecompared with the sensitized MBL-A (+/+) group. Lung TH2 cytokine levels(IL-4, IL-5 and IL-13) were significantly lower in A.fumigatus-sensitized MBL-A(−/−) mice compared to the wild-type group atday 4 after conidia. These results indicate that MBL-A and the lectinpathway have a major role in the development and maintenance of AHRduring chronic fungal asthma.

The findings detailed above suggest the involvement of lectin-dependentcomplement activation in the pathogenesis of asthma. Experimental datasuggest that factor B activation plays a pivotal role. In light of thefundamental role for LEA-1 in the lectin-dependent activation of factorB and subsequent activation of the alternative pathway, it is expectedthat LEA-1 blocking agents will be beneficial for the treatment ofcertain forms of asthma mediated by the alternative pathway. Such atreatment may thus be particularly useful in house dust mite-inducedasthma, or asthma caused by environmental triggers such as cigarettesmoke or diesel exhaust. Asthmatic responses triggered by grass pollenon the other hand are likely to invoke LEA-2-dependent complementactivation. Therefore, LEA-2-blocking agents are expected to beparticularly useful in treating the asthmatic conditions in this subsetof patients.

In view of the data detailed above, the inventors believe that LEA-1 andLEA-2 mediate pathologic complement activation in asthma. Depending onthe inciting allergic agent, LEA-1 or LEA-2 may be preferentiallyinvolved. Thus, a LEA-1-blocking agent combined with a LEA-2-blockingagent may have utility in the treatment of multiple forms of asthmaregardless of the underlying etiology. LEA-1 and LEA-2-blocking agentsmay have complementary, additive or synergistic effects in preventing,treating or reversing pulmonary inflammation and symptoms of asthma.

Combined LEA-1 and LEA-2 inhibition may be accomplished byco-administration of a LEA-1-blocking agent and a LEA2-blocking agent.Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in asingle molecular entity, such as a bispecific antibody composed ofMASP-1/3 and a MASP-2-specific binding site, or a dual specificityantibody where each binding site can bind to and block MASP-1/3 orMASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of asthma, comprisingadministering a composition comprising a therapeutically effectiveamount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent,a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitoryagent, in a pharmaceutical carrier to a subject suffering from, or atrisk for developing asthma. The MASP-1, MASP-3, or MASP-1/3 inhibitorycomposition may be administered to the subject systemically, such as byintra arterial, intravenous, intramuscular, inhalational, nasal,subcutaneous or other parenteral administration, or potentially by oraladministration for non peptidergic agents. Administration may berepeated as determined by a physician until the condition has beenresolved or is controlled.

In one embodiment, the method according to this aspect of the inventionfurther comprises inhibiting LEA-2-dependent complement activation fortreating, preventing, or reducing the severity of asthma, comprisingadministering a therapeutically effective amount of a MASP-2 inhibitoryagent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subjectsuffering from, or at risk for developing asthma. As detailed above, theuse of a combination of pharmacologic agents that individually blockLEA-1 and LEA-2, is expected to provide an improved therapeutic outcomein treating or preventing or reducing the severity of asthma as comparedto the inhibition of LEA-1 alone. This outcome can be achieved forexample, by co-administration of an antibody that has LEA-1-blockingactivity together with an antibody that has LEA-2-blocking activity. Insome embodiments, LEA-1- and LEA-2-blocking activities are combined intoa single molecular entity, and that such entity with combined LEA-1- andLEA-2-blocking activity. Such an entity may comprise or consist of abispecific antibody where one antigen-combining site specificallyrecognizes MASP-1 and blocks LEA-1 and the second antigen-combining sitespecifically recognizes MASP-2 and blocks LEA-2. Alternatively, such anentity may consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes MASP-3 and thus blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Such an entity may optimally consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1while the second antigen-combining site specifically recognized MASP-2and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subjectsystemically, such as by intra arterial, intravenous, intramuscular,inhalational, nasal, subcutaneous or other parenteral administration, orpotentially by oral administration for non peptidergic agents.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and optional MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treating, preventing orreducing the severity of a asthma in a subject suffering from, or atrisk for developing asthma. Alternatively, the composition may beadministered at periodic intervals such as daily, biweekly, weekly,every other week, monthly or bimonthly over an extended period of timefor treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as asthma.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developing asthmacomprising an effective amount of a high affinity monoclonal antibody orantigen binding fragment thereof as disclosed herein that binds to humanMASP-3 and inhibits alternative pathway complement activation to treator reduce the risk of developing asthma, such as, for example, whereinsaid antibody or antigen binding fragment thereof comprises (a) a heavychain variable region comprising (i) VHCDR1 comprising SEQ ID NO:84,(ii) VHCDR2 comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3comprising SEQ ID NO:88; and (b) a light chain variable regioncomprising (i) VLCDR1 comprising SEQ ID NO:142, SEQ ID NO:257, SEQ IDNO:258 or SEQ ID NO:259 (ii) VLCDR2 comprising SEQ ID NO:144 and (iii)VLCDR3 comprising SEQ ID NO:161.

H. The Role of MASP-3 in Dense Deposit Disease, and Therapeutic MethodsUsing MASP-3 Inhibitory Antibodies, Optionally in Combination withMASP-2 Inhibitory Agents

Membranoproliferative glomerulonephritis (MPGN) is a kidney disordercharacterized morphologically by mesangial cell proliferation andthickening of the glomerular capillary wall due to subendothelialextension of the mesangium. MPGN is classified as primary (also referredto as idiopathic) or secondary, with underlying diseases such asinfectious diseases, systemic immune complex diseases, neoplasms,chronic liver disease, and others. Idiopathic MPGN includes threemorphologic types. Type I, or classical MPGN, is characterized bysubendothelial deposits of immune complexes and activation of theclassical complement pathway. Type II, or dense deposit disease (DDD),is characterized by additional intra-membraneous dense deposits. TypeIII is characterized by additional subepithelial deposits. IdiopathicMPGN is rare, accounting for approximately 4 to 7% of primary renalcauses of nephrotic syndrome (Alchi, B. and Jayne, D. Pediatr. Nephrol.25:1409-1418, 2010). MPGN primarily affects children and young adultsand may present as nephrotic syndrome, acute nephritic syndrome,asymptomatic proteinuria and hematuria, or recurrent gross hematuria.Renal dysfunction occurs in the majority of patients and the disease hasa slowly progressive course, with approximately 40% of patientsdeveloping end-stage renal disease within 10 years of diagnosis (Alchiand Jayne, 2010, supra). Current treatment options includecorticosteroids, immunosuppressives, antiplatelet regimens, and plasmaexchange.

DDD is diagnosed by the absence of immunoglobulin and presence of C3 byimmunofluorescence staining of renal biopsies, and electron microscopyshows characteristic dense osmiophilic deposits along the glomerularbasement membranes. DDD is caused by dysregulation of the alternativepathway of complement (Sethi et al, Clin J Am Soc Nephrol. 6(5):1009-17,2011), which can arise from a number of different mechanisms. The mostcommon complement system abnormality in DDD is the presence of C3nephritic factors which are autoantibodies to the alternative pathway C3convertase (C3bBb) that increases its half-life and therefore activationof the pathway (Smith, R. J. H. et al., Mol. Immunol. 48:1604-1610,2011). Other alternative pathway abnormalities include factor Hautoantibody that blocks the function of factor H, gain of function C3mutations, and genetic deficiency of factor H (Smith et al., 2011,supra). Recent case reports show that eclizumab (anti-CS monoclonalantibody) treatment was associated with improvements in renal functionin two patients with DDD (Daina, E. et al., New Engl. J. Med.366:1161-1163, 2012; Vivarelli, M. et al., New Engl. J. Med.366:1163-1165, 2012), suggesting a causative role for complementactivation in renal outcomes.

Given the above genetic, functional and immunohistochemical andanecdotal clinical data, the central role for complement in thepathogenesis of DDD is well established. Thus, interventions that blockthe disease-causing mechanisms of complement activation, or thesubsequent complement activation products, are expected to betherapeutically useful to treat this condition.

While the human genetic data suggest that inappropriate control orexcessive activation of the alternative pathways amplification loopplays a key role, complement-initiating events have not been identified.Immunohistochemical studies in renal biopsies show evidence of MBLdeposition in diseased tissue, suggesting involvement of the lectinpathways in the initiation of pathological complement activation in DDD(Lhotta et al, Nephrol Dial Transplant., 14(4):881-6, 1999). Theimportance of the alternative pathway has been further corroborated inexperimental models. Factor H-deficient mice develop progressiveproteinuria and the renal pathological lesions characteristic of thehuman condition (Pickering et al., Nat Genet., 31(4):424, 2002).Pickering et al. further demonstrated that ablation of factor B, whichmediates LEA-1-dependent activation of the alternative pathway, fullyprotects factor H-deficient mice from DDD (Pickering et al., Nat Genet.,31(4):424, 2002).

Thus it is expected that agents that block LEA-1 will effectively blocklectin-dependent activation of the alternative pathway, and will thusprovide an effective treatment for DDD. Given that the alternativepathway amplification loop is dysregulated in DDD patients, it canfurther be expected that agents that block the amplification loop willbe effective. Since LEA-1-targeting agents that block MASP-1 or MASP-1and MASP-3 inhibit the maturation of factor D, such agents are predictedto effectively block the alternative pathway amplification loop.

As detailed above, pronounced MBL deposition has been found in diseasedrenal specimens, highlighting the probable involvement of lectin-drivenactivation events in DDD pathogenesis. Once an initial tissue injury tothe glomerular capillaries is established, it is likely that additionalMBL binding to injured glomerular endothelium and underlying mesangialstructures occurs. Such tissue injuries are well known to lead toactivation of LEA-2, which can thus cause further complement activation.Therefore, LEA-2-blocking agents are also expected to have utility inpreventing further complement activation on injured glomerularstructures, and thus forestall further disease progression towards endstage renal failure.

The data detailed above suggest that LEA-1 and LEA-2 promote separatepathologic complement activation processes in DDD. Thus, aLEA-1-blocking agent and a LEA-2 blocking agent, either alone or incombination are expected to be useful for treating DDD.

When used in combination, LEA-1- and LEA-2-blocking agents are expectedto be more efficacious than either agent alone, or useful for treatingdifferent stages of the disease. LEA-1- and LEA-2-blocking agents maythus have complementary, additive or synergistic effects in preventing,treating or reversing DDD-associated renal dysfunction.

Combined LEA-1 and LEA-2 inhibition may be accomplished byco-administration of a LEA-1 blocking agent and a LEA2 blocking agent.Optimally, LEA-1 and LEA-2 blocking agents with inhibitory function maybe encompassed in a single molecular entity, such as a bispecificantibody composed of MASP-1/3 and a MASP-2-specific binding site, or adual-specificity antibody where each binding site can bind to and blockMASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of dense depositdisease, comprising administering a composition comprising atherapeutically effective amount of a LEA-1 inhibitory agent comprisinga MASP 1 inhibitory agent, a MASP 3 inhibitory agent, or a combinationof a MASP 1/3 inhibitory agent, in a pharmaceutical carrier to a subjectsuffering from, or at risk for developing dense deposit disease. TheMASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administeredto the subject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependentcomplement activation for treating, preventing, or reducing the severityof dense deposit disease, comprising administering a therapeuticallyeffective amount of a MASP-2 inhibitory agent to a subject sufferingfrom, or at risk for developing dense deposit disease. In anotheraspect, a method is provided comprising inhibiting both LEA-1 andLEA-2-dependent complement activation for treating, preventing, orreducing the severity of dense deposit disease, comprising administeringa therapeutically effective amount of a MASP-2 inhibitory agent and aMASP-1, MASP-3, or MASP-1/3-inhibitory agent to a subject sufferingfrom, or at risk for developing dense deposit disease.

In some embodiments, the method comprises inhibiting bothLEA-1-dependent complement activation and LEA-2-dependent complementactivation. As detailed above, the use of a combination of pharmacologicagents that individually block LEA-1 and LEA-2, is expected to providean improved therapeutic outcome in treating, preventing or reducing theseverity of dense deposit disease as compared to the inhibition of LEA-1alone. This outcome can be achieved for example, by co-administration ofan antibody that has LEA-1-blocking activity together with an antibodythat has LEA-2-blocking activity. In some embodiments, LEA-1- andLEA-2-blocking activities are combined into a single molecular entity,and that such entity with combined LEA-1- and LEA-2-blocking activity.Such an entity may comprise or consist of a bispecific antibody whereone antigen-combining site specifically recognizes MASP-1 and blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Alternatively, such an entity may consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes MASP-3 and thus blocks LEA-1 and the secondantigen-combining site specifically recognizes MASP-2 and blocks LEA-2.Such an entity may optimally consist of a bispecific monoclonal antibodywhere one antigen-combining site specifically recognizes both MASP-1 andMASP-3 and thus blocks LEA-1 while the second antigen-combining sitespecifically recognized MASP-2 and blocks LEA-2.

The LEA-1 and/or LEA-2 inhibitory agents may be administered to thesubject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treating, preventing orreducing the severity of dense deposit disease in a subject in needthereof. Alternatively, the composition may be administered at periodicintervals such as daily, biweekly, weekly, every other week, monthly orbimonthly over an extended period of time for treatment of a subject inneed thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as dense deposit disease.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developing densedeposit disease comprising an effective amount of a high affinitymonoclonal antibody or antigen binding fragment thereof as disclosedherein that binds to human MASP-3 and inhibits alternative pathwaycomplement activation to treat or reduce the risk of developing densedeposit disease, such as, for example, wherein said antibody or antigenbinding fragment thereof comprises (a) a heavy chain variable regioncomprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprisingSEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88;and (b) a light chain variable region comprising (i) VLCDR1 comprisingSEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ ID NO:161.

I. The Role of Masp-3 in PAUCI-immune Necrotizing CrescenticGlomerulonephritis, and Therapeutic Methods Using MASP-3 InhibitoryAntibodies, Optionally in Combination with and MASP-2 Inhibitory Agents

Pauci-immune necrotizing crescentic glomerulonephritis (NCGN) is a formof rapidly progressive glomerulonephritis in which glomerular capillarywalls show signs of inflammation yet have a paucity of detectableimmunocomplex deposition or antibodies against the glomerular basementmembrane. The condition is associated with a rapid decline in renalfunction. Most patients with NCGN are found to have antineutrophilcytoplasmic autoantibodies (ANCA) and thus belong to a group of diseasestermed ANCA-associated vasculitis. Vasculitis is a disorder of bloodvessels characterized by inflammation and fibrinoid necrosis of thevessel wall. Systemic vasculitides are classified based on vessel size:large, medium, and small. Several forms of small-vessel vasculitis areassociated with the presence of ANCA, namely Wegener granulomatosis,microscopic polyangiitis, Churg-Strauss syndrome, and renal-limitedvasculitis (NCGN). They can also be a manifestation of underlyingconditions such as systemic lupus erythematosus. The target antigens forANCA include proteinase-3 (PR3) and myeloperoxidase (MPO). Pauci-immuneNCGN is rare, with a reported incidence of approximately 4 per millionin Wessex, United Kingdom (Hedger, N. et al., Nephrol. Dial. Transplant.15:1593-1599, 2000). In the Wessex series of 128 patients withpauci-immune NCGN, 73% were ANCA-positive and initial dialysis wasrequired by 59% of patients and 36% needed long-term dialysis.Treatments for pauci-immune NCGN include corticosteroids andimmunsuppressive agents such as cyclophosphamide and azathioprine.Additional treatment options for ANCA-associated vasculitides includerituximab and plasma exchange (Chen, M. and Kallenberg, C. G. M. Nat.Rev. Rheumatol. 6:653-664, 2010).

Although NCGN is characterized by a paucity of complement deposition,the alternative pathway of complement has been implicated in itspathogenesis. A renal biopsy evaluation of 7 patients withMPO-ANCA-associated pauci-immune NCGN detected the presence of membraneattack complex, C3d, factor B, and factor P (which were not detected inbiopsies from normal controls or patients with minimal change disease),whereas C4d and mannose binding lectin were not detected, suggestingselective activation of the alternative pathway (Xing, G. Q. et al. J.Clin. Immunol. 29:282-291, 2009). Experimental NCGN can be induced bytransfer of anti-MPO IgG into wild-type mice or anti-MPO splenocytesinto immune-deficient mice (Xiao, H. et al. J. Clin. Invest.110:955-963, 2002). In this mouse model of NCGN, the role of specificcomplement activation pathways was investigated using knockout mice.After injection of anti-MPO IgG, C4−/− mice developed renal diseasecomparable to wild-type mice whereas C5−/− and factor B−/− mice did notdevelop renal disease, indicating that the alternative pathway wasinvolved in this model and the classical and lectin pathways were not(Xiao, H. et al. Am. J. Pathol. 170:52-64, 2007). Moreover, incubationof MPO-ANCA or PR3-ANCA IgG from patients with TNF—primed humanneutrophils caused release of factors that resulted in complementactivation in normal human serum as detected by generation of C3a; thiseffect was not observed with IgG from healthy subjects, suggesting thepotential pathogenic role of ANCA in neutrophil and complementactivation (Xiao et al., 2007, supra).

Based on the role outlined above for the alternative pathway in thiscondition, it is expected that blocking the activation of thealternative pathway will have utility in the treatment of ANCA positiveNCGN. Given the requirement for fB activation for pathogenesis, it isexpected that inhibitors of LEA-1 will be particularly useful intreating this condition, and in preventing the further decline in renalfunction in these patients.

Yet another subset of patients develops progressive renal vasulitis withcrescent formation accompanied by a rapid decline in renal function inthe absence of ANCA. This form of the condition is termed ANCA-negativeNCGN and constitutes about one third of all patients with pauci immuneNCGN (Chen et al, JASN 18(2): 599-605, 2007). These patients tend to beyounger, and renal outcomes tend to be particularly severe. (Chen etal., Nat Rev Nephrol., 5(6):313-8, 2009). A discriminating pathologicalfeature of these patients is the deposition of MBL and C4d in renallesions (Xing et al., J Clin Immunol. 30(1):144-56, 2010). MBL and C4dstaining intensity in renal biopsies correlated negatively with renalfunction (Xing et al., 2010, supra). These findings suggest an importantrole for lectin-dependent complement activation in pathogenesis. Thefact that C4d, but not factor B is commonly found in diseased tissuespecimens indicates LEA-2 involvement.

Based on the role of lectin-dependent complement activation in ANCAnegative NCGN described above, it is expected that blocking theactivation of the LEA-2 pathway will have utility in the treatment ofANCA negative NCGN.

The data detailed above suggest that LEA-1 and LEA-2 mediate pathologiccomplement activation in ANCA-positive and ANCA-negative NCGN,respectively. Thus, a LEA-1-blocking agent combined with aLEA-2-blocking agent is expected to have utility in the treatment of allforms of pauci-immune NCGN, regardless of the underlying etiology.LEA-1- and LEA-2-blocking agents may thus have complementary, additiveor synergistic effects in preventing, treating or reversingNCGN-associated renal dysfunction.

LEA-1 and LEA-2 inhibitors used together may achieve additionaltreatment benefit compared to either agent alone, or may provideeffective treatment for a wider spectrum of patient subsets. CombinedLEA-1 and LEA-2 inhibition may be accomplished by co-administration of aLEA-1 blocking agent and a LEA-2 blocking agent. Optimally, LEA-1 andLEA-2 inhibitory function may be encompassed in a single molecularentity, such as a bispecific antibody composed of MASP-1/3 and aMASP-2-specific binding site, or a dual-specificity antibody where eachbinding site can bind to and block MASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of pauci-immunenecrotizing crescentic glomerulonephritis, comprising administering acomposition comprising a therapeutically effective amount of a LEA-1inhibitory agent comprising a MASP-1 inhibitory agent, a MASP-3inhibitory agent, or a combination of a MASP-1/3 inhibitory agent, in apharmaceutical carrier to a subject suffering from, or at risk fordeveloping pauci-immune necrotizing crescentic glomerulonephritis. TheMASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administeredto the subject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependentcomplement activation for treating, preventing, or reducing the severityof pauci-immune necrotizing crescentic glomerulonephritis, comprisingadministering a therapeutically effective amount of a MASP-2 inhibitoryagent to a subject suffering from, or at risk for developingpauci-immune necrotizing crescentic glomerulonephritis. In anotheraspect, a method is provided comprising inhibiting both LEA-1 andLEA-2-dependent complement activation for treating, preventing, orreducing the severity of pauci-immune necrotizing crescenticglomerulonephritis, comprising administering a therapeutically effectiveamount of a MASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3inhibitory agent to a subject in need thereof.

In some embodiments, the method comprises inhibiting bothLEA-1-dependent complement activation and LEA-2-dependent complementactivation. As detailed above, the use of a combination of pharmacologicagents that individually block LEA-1 and LEA-2, is expected to providean improved therapeutic outcome in treating or preventing or reducingthe severity of pauci-immune necrotizing crescentic glomerulonephritisas compared to the inhibition of LEA-1 alone. This outcome can beachieved for example, by co-administration of an antibody that hasLEA-1-blocking activity together with an antibody that hasLEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blockingactivities are combined into a single molecular entity, and that suchentity with combined LEA-1- and LEA-2-blocking activity. Such an entitymay comprise or consist of a bispecific antibody where oneantigen-combining site specifically recognizes MASP-1 and blocks LEA-1and the second antigen-combining site specifically recognizes MASP-2 andblocks LEA-2. Alternatively, such an entity may consist of a bispecificmonoclonal antibody where one antigen-combining site specificallyrecognizes MASP-3 and thus blocks LEA-1 and the second antigen-combiningsite specifically recognizes MASP-2 and blocks LEA-2. Such an entity mayoptimally consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes both MASP-1 and MASP-3and thus blocks LEA-1 while the second antigen-combining sitespecifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subjectsystemically, such as by intra arterial, intravenous, intramuscular,inhalational, nasal, subcutaneous or other parenteral administration, orpotentially by oral administration for non peptidergic agents.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treating, preventing orreducing the severity of pauci-immune necrotizing crescenticglomerulonephritis. Alternatively, the composition may be administeredat periodic intervals such as daily, biweekly, weekly, every other week,monthly or bimonthly over an extended period of time for treatment of asubject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as Pauci-immune necrotizing crescenticglomerulonephritis (NCGN).

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingPauci-immune necrotizing crescentic glomerulonephritis (NCGN) comprisingan effective amount of a high affinity monoclonal antibody or antigenbinding fragment thereof as disclosed herein that binds to human MASP-3and inhibits alternative pathway complement activation to treat orreduce the risk of developing Pauci-immune necrotizing crescenticglomerulonephritis (NCGN), such as, for example, wherein said antibodyor antigen binding fragment thereof comprises (a) a heavy chain variableregion comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQID NO:88; and (b) a light chain variable region comprising (i) VLCDR1comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259(ii) VLCDR2 comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ IDNO:161.

J. The Role of MASP-3 in Traumatic Brain Injury, and Therapeutic MethodsUsing MASP-3 Inhibitory Antibodies, Optionally in Combination with andMASP-2 Inhibitory Agents

Traumatic brain injury (TBI) is a major global health problem that leadsto at least 10 million deaths or hospitalizations annually (Langlois, J.A. et al., J. Head Trauma Rehabil. 21:375-378, 2006). In 2003 there werean estimated 1.6 million TBIs in the United States, including 1.2million emergency department visits, 290,000 hospitalizations, and51,000 deaths (Rutland-Brown, W. et al., J. Head Trauma Rehabil.21:544-548, 2006). The majority of TBIs in the United States are causedby falls and motor vehicle traffic. TBI can result in long-term orlifelong physical, cognitive, behavioral, and emotional consequences.Over 5 million Americans are living with long-term or lifelongdisability associated with a TBI (Langlois et al., 2006, supra).

TBI may involve penetration of the brain substance (“penetrating”injuries) or injuries that do not penetrate the brain (“closed”injuries). The injury profiles and associated neurobehavioral sequelaecan be quite different between penetrating and closed TBI. Although eachinjury is unique, certain brain regions are particularly vulnerable totrauma-induced damage, including the frontal cortex and subfrontal whitematter, the basal ganglia and diencephalon, the rostral brain stem, andthe temporal lobes including the hippocampi (McAllister, T. W. DialoguesClin. Neurosci. 13:287-300, 2011). TBI can lead to changes in severalneurotransmitter systems, including release of glutamate and otherexcitatory amino acids during the acute phase and chronic alterations inthe catecholaminergic and cholinergic systems, which may be associatedwith neurobehavioral disability (McAllister, 2011, supra). Survivors ofsignificant TBI often suffer from cognitive defects, personalitychanges, and increased psychiatric disorders, particularly depression,anxiety, and post-traumatic stress disorder. Despite intense research,no clinically effective treatment for TBI that can reduce mortality andmorbidity and improve functional outcome has yet to be found.

Complement Factors and TBI

Numerous studies have identified a relationship of complement proteinsand neurological disorders, including Alzheimer's disease, multiplesclerosis, myasthenia gravis, Guillain-Barré syndrome, cerebral lupus,and stroke (reviewed in Wagner, E., et al., Nature Rev Drug Disc. 9:43-56, 2010). Recently a role for C1q and C3 in synapse elimination hasbeen demonstrated, thus complement factors are likely involved in bothnormal CNS function and neurodegenerative disease (Stevens, B. et al.,Cell 131: 1164-1178, 2007). The gene for MASP-1 and MASP-3 isextensively expressed in the brain and also in a glioma cell line, T98G(Kuraya, M. et al., Int Immunol., 15:109-17, 2003), consistent with arole of the lectin pathway in the CNS.

MASP-1 and MASP-3 are key to immediate defense against pathogens andaltered self-cells, but the lectin pathway also is responsible forsevere tissue damage after stroke, heart attack, and other ischemiareperfusion injuries. Similarly, MASP-1 and MASP-3 are likely mediatorsin the tissue damage caused by TBI. Inhibition of Factor B in thealternative pathway has been shown to attenuate TBI in two mouse models.Factor B knockout mice are protected from complement-mediatedneuroinflammation and neuropathology after TBI (Leinhase I, et al., BMCNeurosci. 7:55, 2006). In addition, anti-factor B antibody attenuatedcerebral tissue damage and neuronal cell death in TBI induced mice(Leinhase I, et al., J Neuroinflammation 4:13, 2007). MASP-3 directlyactivates Factor B (Iwaki, D. et al., J Immunol. 187:3751-8, 2011) andtherefore is also a likely mediator in TBI. Similar to inhibition ofFactor B, LEA-1 inhibitors, such as antibodies against MASP-3 areexpected to provide a promising strategy for treating tissue damage andsubsequent sequelae in TBI.

Thus, LEA-1 and LEA-2 inhibitors may have independent therapeuticbenefit in TBI. In addition, LEA-1 and LEA-2 inhibitors used togethermay achieve additional treatment benefit compared to either agent alone,or may provide effective treatment for a wider spectrum of patientsubsets. Combined LEA-1 and LEA-2 inhibition may be accomplished byco-administration of a LEA-1-blocking agent and a LEA2-blocking agent.Optimally, LEA-1 and LEA-2 inhibitory function may be encompassed in asingle molecular entity, such as a bispecific antibody composed ofMASP-1/3 and a MASP-2-specific binding site, or a dual-specificityantibody where each binding site can bind to and block MASP-1/3 orMASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, or reducing the severity of traumatic brain injury,comprising administering a composition comprising a therapeuticallyeffective amount of a LEA-1 inhibitory agent comprising a MASP-1inhibitory agent, a MASP-3 inhibitory agent, or a combination of aMASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subjectsuffering from a traumatic brain injury. The MASP-1, MASP-3, or MASP-1/3inhibitory composition may be administered to the subject systemically,such as by intra arterial, intravenous, intramuscular, inhalational,nasal, intracranial, subcutaneous or other parenteral administration, orpotentially by oral administration for non peptidergic agents.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependentcomplement activation for treating, or reducing the severity oftraumatic brain injury, comprising administering a therapeuticallyeffective amount of a MASP-2 inhibitory agent to a subject sufferingfrom a traumatic brain injury. In another aspect, a method is providedcomprising inhibiting both LEA-1 and LEA-2-dependent complementactivation for treating, or reducing the severity of traumatic braininjury, comprising administering a therapeutically effective amount of aMASP-2 inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitoryagent to a subject suffering from a traumatic brain injury.

In some embodiments, the method comprises inhibiting bothLEA-1-dependent complement activation and LEA-2-dependent complementactivation. As detailed above, the use of a combination of pharmacologicagents that individually block LEA-1 and LEA-2 is expected to provide animproved therapeutic outcome in treating or reducing the severity oftraumatic brain injury as compared to the inhibition of LEA-1 alone.This outcome can be achieved for example, by co-administration of anantibody that has LEA-1-blocking activity together with an antibody thathas LEA-2-blocking activity. In some embodiments, LEA-1- andLEA-2-blocking activities are combined into a single molecular entity,and that such entity with combined LEA-1- and LEA-2-blocking activity.Such an entity may comprise or consist of a bispecific antibody whereone antigen-combining site specifically recognizes MASP-1 and blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Alternatively, such an entity may consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes MASP-3 and thus blocks LEA-1 and the secondantigen-combining site specifically recognizes MASP-2 and blocks LEA-2.Such an entity may optimally consist of a bispecific monoclonal antibodywhere one antigen-combining site specifically recognizes both MASP-1 andMASP-3 and thus blocks LEA-1 while the second antigen-combining sitespecifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subjectsystemically, such as by intra arterial, intravenous, intramuscular,inhalational, nasal, subcutaneous, intracranial, or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treating or reducing theseverity of traumatic brain injury. Alternatively, the composition maybe administered at periodic intervals such as daily, biweekly, weekly,every other week, monthly or bimonthly over an extended period of timefor treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as traumatic brain injury.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingtraumatic brain injury comprising an effective amount of a high affinitymonoclonal antibody or antigen binding fragment thereof as disclosedherein that binds to human MASP-3 and inhibits alternative pathwaycomplement activation to treat or reduce the risk of developingtraumatic brain injury, such as, for example, wherein said antibody orantigen binding fragment thereof comprises (a) a heavy chain variableregion comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQID NO:88; and (b) a light chain variable region comprising (i) VLCDR1comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259(ii) VLCDR2 comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ IDNO:161.

K. The Role of MASP-3 in Aspiration Pneumonia, and Therapeutic MethodsUsing MASP-3 Inhibitory Antibodies, Optionally in Combination withMasp-2 Inhibitory Agents

Aspiration is defined as the inhalation of either oropharyngeal orgastric contents into the lower airways. Aspiration may result incomplications of aspiration (chemical) pneumonitis, primary bacterialaspiration pneumonia, or secondary bacterial infection of chemicalpneumonitis. Risk factors for aspiration include decreased levels ofconsciousness (e.g., head trauma, alcohol or drug-induced alterations insensorium, stroke), various gastrointestinal and esophagealabnormalities, and neuromuscular diseases. It is estimated that 5-15% ofthe 4.5 million cases of community-acquired pneumonia are due toaspiration pneumonia (Marik, P. E. New Engl. J. Med. 344:665-671, 2001).Treatment of chemical pneumonitis is mainly supportive and the use ofempiric antibiotics is controversial. Treatment of bacterial aspirationpneumonia is with appropriate antibiotics, which is based on whether theaspiration occurred in the community or in the hospital as the likelycausative organisms differ between these settings. Measures should betaken to prevent aspiration in high-risk patients, for example elderlypatients in nursing homes who have impaired gag reflexes. Measures thathave been shown to be effective prophylaxis include elevation of thehead of the bed while feeding, dental prophylaxis, and good oralhygiene. Prophylactic antibiotics have not been shown to be effectiveand are discouraged as they may lead to the emergence of resistantorganisms.

Modulation of complement components has been proposed for numerousclinical indications, including infectious disease—sepsis, viral,bacterial, and fungal infections—and pulmonary conditions—respiratorydistress syndrome, chronic obstructive pulmonary disease, and cysticfibrosis (reviewed in Wagner, E., et al., Nature Rev Drug Disc. 9:43-56, 2010). Support for this proposal is provided by numerous clinicaland genetic studies. For example, there is a significantly decreasedfrequency of patients with low MBL levels with clinical tuberculosis(Soborg et al., Journal of Infectious Diseases 188:777-82, 2003),suggesting that low levels of MBL are associated with protection fromdisease.

In a murine model of acid aspiration injury, Weiser M R et al., J. Appl.Physiol. 83(4): 1090-1095, 1997, demonstrated that C3-knockout mice wereprotected from serious injury; whereas C4-knockout mice were notprotected, indicating that complement activation is mediated by thealternative pathway. Consequently, blocking the alternative pathway withLEA-1 inhibitors is expected to provide a therapeutic benefit inaspiration pneumonia.

Thus, LEA-1 and LEA-2 inhibitors may have independent therapeuticbenefit in aspiration pneumonia. In addition, LEA-1 and LEA-2 inhibitorsused together may achieve additional treatment benefit compared toeither agent alone, or may provide effective treatment for a widerspectrum of patient subsets. Combined LEA-1 and LEA-2 inhibition may beaccomplished by co-administration of a LEA-1-blocking agent and aLEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function maybe encompassed in a single molecular entity, such as a bi-specificantibody composed of MASP-1/3 and a MASP-2-specific binding site, or adual-specificity antibody where each binding site binds to and blocksMASP-1/3 or MASP-2.

An aspect of the invention thus provides a method for inhibiting LEA-1dependent complement activation to treat aspiration pneumonia byadministering a composition comprising a therapeutically effectiveamount of a MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or acombination of a MASP-1/3 inhibitory agent, in a pharmaceutical carrierto a subject suffering from such a condition or other complementmediated pneumonia. The MASP-1, MASP-3, or MASP-1/3 inhibitorycomposition may be administered locally to the lung, as by an inhaler.Alternately, the MASP-1, MASP-3, or MASP-1/3 inhibitory agent may beadministered to the subject systemically, such as by intra arterial,intravenous, intramuscular, inhalational, nasal, subcutaneous or otherparenteral administration, or potentially by oral administration.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing or reducing the severity of aspirationpneumonia, comprising administering a composition comprising atherapeutically effective amount of a LEA-1 inhibitory agent comprisinga MASP-1 inhibitory agent, a MASP-3 inhibitory agent, or a combinationof a MASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subjectsuffering from, or at risk for developing aspiration pneumonia. TheMASP-1, MASP-3, or MASP-1/3 inhibitory composition may be administeredto the subject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependentcomplement activation for treating, preventing or reducing the severityof aspiration pneumonia, comprising administering a therapeuticallyeffective amount of a MASP-2 inhibitory agent to a subject sufferingfrom, or at risk for developing aspiration pneumonia. In another aspect,a method is provided comprising inhibiting both LEA-1 andLEA-2-dependent complement activation for treating, or reducing theseverity of aspiration pneumonia, comprising administering atherapeutically effective amount of a MASP-2 inhibitory agent and aMASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering fromaspiration pneumonia.In some embodiments, the method comprisesinhibiting both LEA-1-dependent complement activation andLEA-2-dependent complement activation. As detailed above, the use of acombination of pharmacologic agents that individually block LEA-1 andLEA-2, is expected to provide an improved therapeutic outcome intreating or reducing the severity of aspiration pneumonia as compared tothe inhibition of LEA-1 alone. This outcome can be achieved for example,by co-administration of an antibody that has LEA-1-blocking activitytogether with an antibody that has LEA-2-blocking activity. In someembodiments, LEA-1- and LEA-2-blocking activities are combined into asingle molecular entity, and that such entity with combined LEA-1- andLEA-2-blocking activity. Such an entity may comprise or consist of abispecific antibody where one antigen-combining site specificallyrecognizes MASP-1 and blocks LEA-1 and the second antigen-combining sitespecifically recognizes MASP-2 and blocks LEA-2. Alternatively, such anentity may consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes MASP-3 and thus blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Such an entity may optimally consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes both MASP-1 and MASP-3 and thus blocks LEA-1while the second antigen-combining site specifically recognized MASP-2and blocks LEA-2.

The MASP-2 inhibitory agent may be administered to the subjectsystemically, such as by intra arterial, intravenous, intramuscular,inhalational, nasal, subcutaneous, or other parenteral administration,or potentially by oral administration for non-peptidergic agents.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific ordual-inhibitory agents, or co-administration of separate compositions),or a limited sequence of administrations, for treating, preventing orreducing the severity of aspiration pneumonia in a subject in needthereof Alternatively, the composition may be administered at periodicintervals such as daily, biweekly, weekly, every other week, monthly orbimonthly over an extended period of time for treatment of a subject inneed thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as aspiration pneumonia.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingaspiration pneumonia comprising an effective amount of a high affinitymonoclonal antibody or antigen binding fragment thereof as disclosedherein that binds to human MASP-3 and inhibits alternative pathwaycomplement activation to treat or reduce the risk of developingaspiration pneumonia, such as, for example, wherein said antibody orantigen binding fragment thereof comprises (a) a heavy chain variableregion comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQID NO:88; and (b) a light chain variable region comprising (i) VLCDR1comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259(ii) VLCDR2 comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ IDNO:161.

L. The Role Of Masp-3 In Endophthalmitis, and Therapeutic Methods UsingMASP-3 Inhibitory Antibodies, Optionally in Combination with And Masp-2Inhibitory Agents

Endophthalmitis is an inflammatory condition of the intraocular cavitiesand is usually caused by infection. Endophthalmitis may be endogeneous,resulting from hematogenous spread of organisms from a distant source ofinfection (e.g., endocarditis), or exogeneous, from direct inoculationof an organism from the outside as a complication of ocular surgery,foreign bodies, and/or blunt or penetrating ocular trauma. Exogeneousendophthalmitis is much more common than endogenous and most cases ofexogeneous endophthalmitis occur following ocular surgery. In the UnitedStates, cataract surgery is the leading cause of endophthalmitis andoccurs in 0.1-0.3% of this procedure, with an apparent increase in theincidence over the last decade (Taban, M. et al., Arch. Ophthalmol.123:613-620, 2005). Post-surgical endophthalmitis may present eitheracutely, within 2 weeks of surgery, or delayed, months after surgery.Acute endophthalmitis typically presents with pain, redness, lidswelling, and decreased visual acuity. Delayed-onset endophthalmitis isless common than the acute form and patients may report only mild painand photosensitivity. Treatment of endophthalmitis depends on theunderlying cause and may include systemic and/or intravitrealantibiotics. Endophthalmitis may result in decreased or loss of vision.

As previously described for AMD, multiple complement pathway genes havebeen associated with ophthalmologic disorders, and these specificallyinclude genes of the lectin pathway. For example, MBL2 has beenidentified with subtypes of AMD (Dinu V, et al., Genet Epidemiol 31:224-37, 2007). The LEA-1 and LEA-2 pathways are likely to be involved inocular inflammatory conditions such as endophthalmitis (Chow S P et al.,Clin Experiment Ophthalmol. 39:871-7, 2011). Chow et al. examined MBLlevels of patients with endophthalmitis and demonstrated that both MBLlevels and functional lectin pathway activity are significantly elevatedin inflamed human eyes but virtually undetectable in non-inflamedcontrol eyes. This suggests a role for MBL and the lectin pathway insight-threatening ocular inflammatory conditions, particularlyendophthalmitis. Furthermore, in a murine model of corneal fungalkeratitis, the MBL-A gene was one of five upregulated inflammatorypathway genes (Wang Y., et al., Mol Vis 13: 1226-33, 2007).

Thus, LEA-1 and LEA-2 inhibitors are expected to have independenttherapeutic benefit in treating endophthalmitis. In addition, LEA-1 andLEA-2 inhibitors used together may achieve additional treatment benefitcompared to either agent alone, or may provide effective treatment for awider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibitionmay be accomplished by co-administration of a LEA-1-blocking agent and aLEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function maybe encompassed in a single molecular entity, such as a bi-specificantibody composed of MASP-1/3 and a MASP-2-specific binding site, or adual-specificity antibody where each binding site binds to and blocksMASP-1/3 or MASP-2

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of endophthalmitis,comprising administering a composition comprising a therapeuticallyeffective amount of a LEA-1 inhibitory agent comprising a MASP-1inhibitory agent, a MASP-3 inhibitory agent, or a combination of aMASP-1/3 inhibitory agent, in a pharmaceutical carrier to a subjectsuffering from, or at risk for developing endophthalmitis. The MASP-1,MASP-3, or MASP-1/3 inhibitory composition may be administered locallyto the eye, such as by irrigation or application of the composition inthe form of a topical gel, salve or drops, or by intravitrealadministration. Alternately, the MASP-1, MASP-3, or MASP-1/3 inhibitoryagent may be administered to the subject systemically, such as by intraarterial, intravenous, intramuscular, inhalational, nasal, subcutaneousor other parenteral administration, or potentially by oraladministration for non peptidergic agents. Administration may berepeated as determined by a physician until the condition has beenresolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependentcomplement activation for treating, preventing, or reducing the severityof endophthalmitis, comprising administering a therapeutically effectiveamount of a MASP-2 inhibitory agent to a subject suffering from, or atrisk for developing endophthalmitis. In another aspect, a method isprovided comprising inhibiting both LEA-1 and LEA-2-dependent complementactivation for treating, or reducing the severity of endophthalmitis,comprising administering a therapeutically effective amount of a MASP-2inhibitory agent and a MASP-1, MASP-3, or MASP-1/3 inhibitory agent to asubject suffering from endophthalmitis.

In some embodiments, the method comprises inhibiting bothLEA-1-dependent complement activation and LEA-2-dependent complementactivation. As detailed above, the use of a combination of pharmacologicagents that individually block LEA-1 and LEA-2 is expected to provide animproved therapeutic outcome in treating or preventing or reducing theseverity of endophthalmitis, as compared to the inhibition of LEA-1alone. This outcome can be achieved for example, by co-administration ofan antibody that has LEA-1-blocking activity together with an antibodythat has LEA-2-blocking activity. In some embodiments, LEA-1- andLEA-2-blocking activities are combined into a single molecular entity,and that such entity with combined LEA-1- and LEA-2-blocking activity.Such an entity may comprise or consist of a bispecific antibody whereone antigen-combining site specifically recognizes MASP-1 and blocksLEA-1 and the second antigen-combining site specifically recognizesMASP-2 and blocks LEA-2. Alternatively, such an entity may consist of abispecific monoclonal antibody where one antigen-combining sitespecifically recognizes MASP-3 and thus blocks LEA-1 and the secondantigen-combining site specifically recognizes MASP-2 and blocks LEA-2.Such an entity may optimally consist of a bispecific monoclonal antibodywhere one antigen-combining site specifically recognizes both MASP-1 andMASP-3 and thus blocks LEA-1 while the second antigen-combining sitespecifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered locally to the eye, suchas by irrigation or application of the composition in the form of atopical gel, salve or drops, or by intravitreal injection. Alternately,the MASP-2 inhibitory agent may be administered to the subjectsystemically, such as by intra arterial, intravenous, intramuscular,inhalational, nasal, subcutaneous or other parenteral administration, orpotentially by oral administration for non peptidergic agents.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treating, preventing orreducing the severity of endophthalmitis in a subject in need thereof.Alternatively, the composition may be administered at periodic intervalssuch as daily, biweekly, weekly, every other week, monthly or bimonthlyover an extended period of time for treatment of a subject in needthereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as endophthalmitis.

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingendophthalmitis comprising an effective amount of a high affinitymonoclonal antibody or antigen binding fragment thereof as disclosedherein that binds to human MASP-3 and inhibits alternative pathwaycomplement activation to treat or reduce the risk of developingendophthalmitis, such as, for example, wherein said antibody or antigenbinding fragment thereof comprises (a) a heavy chain variable regioncomprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprisingSEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88;and (b) a light chain variable region comprising (i) VLCDR1 comprisingSEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ ID NO:161.

M. The Role Of Masp-3 in Neuromyelitis Optica, and Therapeutic MethodsUsing Masp-3 Inhibitory Antibodies, Optionally in Combination withMasp-2 Inhibitory Agents

Neuromyelitis optica (NMO) is an autoimmune disease that targets theoptic nerves and spinal cord. This results in inflammation of the opticnerve, known as optic neuritis, and the spinal cord, known as myelitis.Spinal cord lesions in NMO may lead to weakness or paralysis in the legsor arms, blindness, bladder and bowel dysfunction, and sensorydysfunction.

NMO shares several similarities to multiple sclerosis (MS), since bothare due to immune attack of CNS targets and both result in demyelination(Papadopoulos and Verkman, Lancet Neurol., 11(6):535-44, 2013). However,the molecular targets, treatments, and lesions for NMO are distinct fromthose of MS. While MS is largely mediated by T cells, NMO patientstypically have antibodies that target the water channel proteinaquaporin 4 (AQP4), a protein found in astrocytes that surround theblood-brain barrier. Interferon beta is the most commonly used therapyfor MS, but it is generally acknowledged to be harmful in NMO. Theinflammatory lesions of NMO are found in the spinal cord and optic nerveand may progress to the brain, including white and gray matter. Thedemyelination that occurs in NMO lesions is mediated by complement(Papadopoulos and Verkman, Lancet Neurol., 11(6):535-44, 2013).

Complement-dependent cytotoxicity appears to be the major mechanismcausing development of NMO. Over 90% of NMO patients have IgG antibodiesagainst AQP4 (Jarius and Wildemann, Jarius S, Wildemann B., Nat RevNeurol. 2010 July; 6(7):383-92). These antibodies initiate formation ofa lesion at the blood brain barrier. The initial antigen-antibodycomplex—AQP4/AQP4-IgG—on the surface of astrocytes activates theclassical pathway of complement. This results in formation of themembrane attack complex on the astrocyte surface, leading to granulocyteinfiltration, demyelination, and ultimately necrosis of astrocytes,oligodendrocytes and neurons (Misu et al., Acta Neuropathol125(6):815-27, 2013). These cellular events are reflected in tissuedestruction and formation of cystic, necrotic lesions.

The classical pathway of complement clearly is critical for NMOpathogenesis. NMO lesions show a vasculocentric deposition ofimmunoglobulin and activated complement components (Jarius et al., NatClin Pract Neurol. 4(4):202-14, 2008). In addition, complement proteinssuch as C5a have been isolated from cerebrospinal fluid of NMO patients(Kuroda et al., J Neuroimmunol.,254(1-2):178-82, 2013). Furthermore,serum IgG obtained from NMO patients can cause complement-dependentcytotoxicity in a mouse NMO model (Saadoun et al., Brain, 133(Pt2):349-61, 2010). A monoclonal antibody against C1q prevents thecomplement mediated destruction of astrocytes and lesions in a mousemodel of NMO (Phuan et al., Acta Neuropathol, 125(6):829-40, 2013).

The alternative pathway of complement serves to amplify overallcomplement activity. Harboe and colleagues (2004) demonstrated thatselective blockade of the alternative pathway inhibited more than 80% ofmembrane attack complex formation induced by the classical pathway(Harboe et al., Clin Exp Immunol 138(3):439-46, 2004). Tüzün andcolleagues (2013) examined both classical and alternative pathwayproducts in NMO patients (Tüzün E, et al.,J Neuroimmunol. 233(1-2):211-5, 2011). C4d, the breakdown product of C4, was measured to evaluateclassical pathway activity and was increased in NMO patient seracompared to controls (an elevation of 2.14-fold). In addition, anincrease of Factor Bb, the breakdown product of the alternative pathwayFactor B, was observed in NMO patients compared to MS patients or normalcontrol individuals (an elevation of 1.33-fold). This suggests thatalternative pathway function is also increased in NMO. This activationwould be expected to increase overall complement activation, and in factsC5b-9, the final product of the complement cascade, was significantlyincreased (a 4.14-fold elevation).

Specific inhibitors of MASP-3 are expected to provide benefit intreating patients suffering from NMO. As demonstrated herein, serumlacking MASP-3 is unable to activate Factor B, an essential component ofC5 convertase, or Factor D, the central activator of the alternativepathway. Therefore, blocking MASP-3 activity with an inhibitory agentsuch as an antibody or small molecule would also be expected to inhibitactivation of Factor B and Factor D. Inhibition of these two factorswill arrest the amplification of the alternative pathway, 0resulting indiminished overall complement activity. MASP-3 inhibition should thussignificantly improve therapeutic outcomes in NMO.

Thus, LEA-1 and/or LEA-2 inhibitors are expected to have independenttherapeutic benefit in treating NMO. In addition, LEA-1 and LEA-2inhibitors used together may achieve additional treatment benefitcompared to either agent alone, or may provide effective treatment for awider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibitionmay be accomplished by co-administration of a LEA-1-blocking agent and aLEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function maybe encompassed in a single molecular entity, such as a bi-specificantibody composed of MASP-1/3 and a MASP-2-specific binding site, or adual-specificity antibody where each binding site binds to and blocksMASP-1/3 or MASP-2

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of NMO, comprisingadministering a composition comprising a therapeutically effectiveamount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent,a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitoryagent, in a pharmaceutical carrier to a subject suffering from, or atrisk for developing NMO. The MASP-1, MASP-3, or MASP-1/3 inhibitorycomposition may be administered locally to the eye, such as byirrigation or application of the composition in the form of a topicalgel, salve or drops, or by intravitreal administration. Alternately, theMASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to thesubject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependentcomplement activation for treating, preventing, or reducing the severityof NMO, comprising administering a therapeutically effective amount of aMASP-2 inhibitory agent to a subject suffering from, or at risk fordeveloping NMO. In another aspect, a method is provided comprisinginhibiting both LEA-1 and LEA-2-dependent complement activation fortreating, or reducing the severity of NMO, comprising administering atherapeutically effective amount of a MASP-2 inhibitory agent and aMASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering fromNMO.

In some embodiments, the method comprises inhibiting bothLEA-1-dependent complement activation and LEA-2-dependent complementactivation. As detailed above, the use of a combination of pharmacologicagents that individually block LEA-1 and LEA-2 is expected to provide animproved therapeutic outcome in treating or preventing or reducing theseverity of NMO, as compared to the inhibition of LEA-1 alone. Thisoutcome can be achieved for example, by co-administration of an antibodythat has LEA-1-blocking activity together with an antibody that hasLEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blockingactivities are combined into a single molecular entity, and that suchentity with combined LEA-1- and LEA-2-blocking activity. Such an entitymay comprise or consist of a bispecific antibody where oneantigen-combining site specifically recognizes MASP-1 and blocks LEA-1and the second antigen-combining site specifically recognizes MASP-2 andblocks LEA-2. Alternatively, such an entity may consist of a bispecificmonoclonal antibody where one antigen-combining site specificallyrecognizes MASP-3 and thus blocks LEA-1 and the second antigen-combiningsite specifically recognizes MASP-2 and blocks LEA-2. Such an entity mayoptimally consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes both MASP-1 and MASP-3and thus blocks LEA-1 while the second antigen-combining sitespecifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered locally to the eye, suchas by irrigation or application of the composition in the form of atopical gel, salve or drops, or by intravitreal injection. Alternately,the MASP-2 inhibitory agent may be administered to the subjectsystemically, such as by intra arterial, intravenous, intramuscular,inhalational, nasal, subcutaneous or other parenteral administration, orpotentially by oral administration for non peptidergic agents.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP 2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treating, preventing orreducing the severity of NMO in a subject in need thereof.Alternatively, the composition may be administered at periodic intervalssuch as daily, biweekly, weekly, every other week, monthly or bimonthlyover an extended period of time for treatment of a subject in needthereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as neuromyelitis optica (NMO).

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingneuromyelitis optica (NMO). comprising an effective amount of a highaffinity monoclonal antibody or antigen binding fragment thereof asdisclosed herein that binds to human MASP-3 and inhibits alternativepathway complement activation to treat or reduce the risk of developingneuromyelitis optica (NMO), such as, for example, wherein said antibodyor antigen binding fragment thereof comprises (a) a heavy chain variableregion comprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2comprising SEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQID NO:88; and (b) a light chain variable region comprising (i) VLCDR1comprising SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259(ii) VLCDR2 comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ IDNO:161.

N. The Role of Masp-3 in Behcet'S Disease, and Therapeutic Methods UsingMasp-3 Inhibitory Antibodies, Optionally in Combination with Masp-2Inhibitory Agents

Behçet's disease, or Behçet's syndrome, is a rare, immune-mediatedsmall-vessel systemic vasculitis that often presents with mucousmembrane ulceration and ocular problems. Behçet's disease (BD) was namedin 1937 after the Turkish dermatologist Hulusi Behçet, who firstdescribed the triple-symptom complex of recurrent oral ulcers, genitalulcers, and uveitis. BD is a systemic, relapsing inflammatory disorderof unknown cause. The inflammatory perivasculitis of BD may involve thegastrointestinal tract, pulmonary, musculoskeletal, cardiovascular, andneurological systems. BD can be fatal due to ruptured vascular aneurysmsor severe neurological complications. Optic neuropathy and atrophy mayresult from vasculitis and occlusion of the vessels supplying the opticnerve. See Al-Araji A, et al., Lancet Neurol., 8(2):192-204, 2009.

The highest incidence of BD is in the Middle East and Far East regions,but it is rare in Europe and North America. BD is often initiallycontrolled with corticosteroids and immunosuppressants, but many casesare refractory with serious morbidity and mortality. Biologic agents,including interferon-alpha, IVIG, anti-TNF, anti-IL-6, and anti-CD20,have shown benefit in some cases, but there is no consensus on besttreatment.

While BD is clearly an inflammatory disorder, its pathobiology is notclear. There are genetic associations with HLA antigens, and genome wideassociation studies have implicated numerous cytokine genes (Kirino etal., Nat Genet, 45(2):202-7, 2013). The hyperactivity of the immunesystem appears to be regulated by the complement system. Increasedlevels of C3 have been observed in BD patient sera (Bardak and Aridoğan,Ocul Immunol Inflamm 12(1):53-8, 2004), and elevated C3 and C4 in thecerebrospinal fluid correlates with disease (Jongen et al., Arch Neurol,49(10):1075-8, 1992).

Tüzün and colleagues (2013) examined both classical and alternativepathway products in sera of BD patients (Tüzün E, et al., JNeuroimmunol, 233(1-2):211-5, 2011). 4d, the breakdown product of C4, isgenerated upstream of the alternative pathway and was measured toevaluate initial classical pathway activity. C4d was increased in BDpatient sera compared to controls (an elevation of 2.18-fold). Factor Bbis the breakdown product of Factor B, and was measured to determineactivity of the alternative pathway. BD patients had an increase offactor Bb compared to normal control individuals (an elevation of2.19-fold) consistent with an increase in BD alternative pathwayfunction. Because the alternative pathway of complement serves toamplify overall complement activity, this activation would be expectedto increase overall complement activation. Harboe and colleagues (2004)demonstrated that selective blockade of the alternative pathwayinhibited more than 80% of membrane attack complex formation induced bythe classical pathway (Harboe M, et al., Clin Exp Immunol,138(3):439-46, 2004). In fact, sC5b-9, the final product of thecomplement cascade, was significantly increased in BD patients (a5.46-fold elevation). Specific inhibitors of MASP-3 should providebenefit in BD. Blocking MASP-3 should inhibit activation of Factor B andFactor D. This will stop the amplification of the alternative pathway,resulting in a diminished response of overall complement activity.MASP-3 inhibition should thus significantly improve therapeutic outcomesin BD. Thus, LEA-1 and/or LEA-2 inhibitors are expected to haveindependent therapeutic benefit in treating BD. In addition, LEA-1 andLEA-2 inhibitors used together may achieve additional treatment benefitcompared to either agent alone, or may provide effective treatment for awider spectrum of patient subsets. Combined LEA-1 and LEA-2 inhibitionmay be accomplished by co-administration of a LEA-1-blocking agent and aLEA-2-blocking agent. Optimally, LEA-1 and LEA-2 inhibitory function maybe encompassed in a single molecular entity, such as a bi-specificantibody composed of MASP-1/3 and a MASP-2-specific binding site, or adual-specificity antibody where each binding site binds to and blocksMASP-1/3 or MASP-2.

In accordance with the foregoing, an aspect of the invention thusprovides a method for inhibiting LEA-1 dependent complement activationfor treating, preventing, or reducing the severity of BD, comprisingadministering a composition comprising a therapeutically effectiveamount of a LEA-1 inhibitory agent comprising a MASP-1 inhibitory agent,a MASP-3 inhibitory agent, or a combination of a MASP-1/3 inhibitoryagent, in a pharmaceutical carrier to a subject suffering from, or atrisk for developing BD. The MASP-1, MASP-3, or MASP-1/3 inhibitorycomposition may be administered locally to the eye, such as byirrigation or application of the composition in the form of a topicalgel, salve or drops, or by intravitreal administration. Alternately, theMASP-1, MASP-3, or MASP-1/3 inhibitory agent may be administered to thesubject systemically, such as by intra arterial, intravenous,intramuscular, inhalational, nasal, subcutaneous or other parenteraladministration, or potentially by oral administration for nonpeptidergic agents. Administration may be repeated as determined by aphysician until the condition has been resolved or is controlled.

In another aspect, a method is provided for inhibiting LEA-2-dependentcomplement activation for treating, preventing, or reducing the severityof BD, comprising administering a therapeutically effective amount of aMASP-2 inhibitory agent to a subject suffering from, or at risk fordeveloping BD. In another aspect, a method is provided comprisinginhibiting both LEA-1 and LEA-2-dependent complement activation fortreating, or reducing the severity of BD, comprising administering atherapeutically effective amount of a MASP-2 inhibitory agent and aMASP-1, MASP-3, or MASP-1/3 inhibitory agent to a subject suffering fromBD.

In some embodiments, the method comprises inhibiting bothLEA-1-dependent complement activation and LEA-2-dependent complementactivation. As detailed above, the use of a combination of pharmacologicagents that individually block LEA-1 and LEA-2 is expected to provide animproved therapeutic outcome in treating or preventing or reducing theseverity of BD, as compared to the inhibition of LEA-1 alone. Thisoutcome can be achieved for example, by co-administration of an antibodythat has LEA-1-blocking activity together with an antibody that hasLEA-2-blocking activity. In some embodiments, LEA-1- and LEA-2-blockingactivities are combined into a single molecular entity, and that suchentity with combined LEA-1- and LEA-2-blocking activity. Such an entitymay comprise or consist of a bispecific antibody where oneantigen-combining site specifically recognizes MASP-1 and blocks LEA-1and the second antigen-combining site specifically recognizes MASP-2 andblocks LEA-2. Alternatively, such an entity may consist of a bispecificmonoclonal antibody where one antigen-combining site specificallyrecognizes MASP-3 and thus blocks LEA-1 and the second antigen-combiningsite specifically recognizes MASP-2 and blocks LEA-2. Such an entity mayoptimally consist of a bispecific monoclonal antibody where oneantigen-combining site specifically recognizes both MASP-1 and MASP-3and thus blocks LEA-1 while the second antigen-combining sitespecifically recognized MASP-2 and blocks LEA-2.

The MASP-2 inhibitory agent may be administered locally to the eye, suchas by irrigation or application of the composition in the form of atopical gel, salve or drops, or by intravitreal injection. Alternately,the MASP-2 inhibitory agent may be administered to the subjectsystemically, such as by intra arterial, intravenous, intramuscular,inhalational, nasal, subcutaneous or other parenteral administration, orpotentially by oral administration for non peptidergic agents.Administration may be repeated as determined by a physician until thecondition has been resolved or is controlled.

Application of the MASP-3 inhibitory compositions and/or the MASP-2inhibitory compositions of the present invention may be carried out by asingle administration of the composition (e.g., a single compositioncomprising MASP-2 and/or MASP-3 inhibitory agents, or bispecific or dualinhibitory agents, or co-administration of separate compositions), or alimited sequence of administrations, for treating, preventing orreducing the severity of BD in a subject in need thereof Alternatively,the composition may be administered at periodic intervals such as daily,biweekly, weekly, every other week, monthly or bimonthly over anextended period of time for treatment of a subject in need thereof.

As described in Examples 11-21 herein, high affinity MASP-3 inhibitoryantibodies have been generated which have therapeutic utility forinhibition of the alternative pathway in AP-related diseases orconditions, such as Behcet's disease (BD).

Accordingly, in one embodiment, the present invention provides a methodfor treating a subject suffering from, or at risk for developingBehcet's disease (BD) comprising an effective amount of a high affinitymonoclonal antibody or antigen binding fragment thereof as disclosedherein that binds to human MASP-3 and inhibits alternative pathwaycomplement activation to treat or reduce the risk of developing Behcet'sdisease (BD), such as, for example, wherein said antibody or antigenbinding fragment thereof comprises (a) a heavy chain variable regioncomprising (i) VHCDR1 comprising SEQ ID NO:84, (ii) VHCDR2 comprisingSEQ ID NO:86 or SEQ ID NO:275 and (iii) VHCDR3 comprising SEQ ID NO:88;and (b) a light chain variable region comprising (i) VLCDR1 comprisingSEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ ID NO:259 (ii) VLCDR2comprising SEQ ID NO:144 and (iii) VLCDR3 comprising SEQ ID NO:161.

MASP-3 Inhibitory Agents

With the recognition that the lectin pathway of complement is composedof two major complement activation arms, LEA-1 and LEA-2, and that therealso is a lectin-independent complement activation arm, comes therealization that it would be highly desirable to specifically inhibitone or more of these effector arms that cause a pathology associatedwithalternative pathway complement activation, such as at least one ofparoxysmal nocturnal hemoglobinuria (PNH), age-related maculardegeneration (AMD, including wet and dry AMD), ischemia-reperfusioninjury, arthritis, disseminated intravascular coagulation, thromboticmicroangiopathy (including hemolytic uremic syndrome (HUS), atypicalhemolytic uremic syndrome (aHUS), thrombotic thrombocytopenic purpura(TTP) or transplant-associated TMA), asthma, dense deposit disease,pauci-immune necrotizing crescentic glomerulonephritis, traumatic braininjury, aspiration pneumonia, endophthalmitis, neuromyelitis optica,Behcet's disease, multiple sclerosis (MS), Guillain Barre Syndrome,Alzheimer's disease, Amylotrophic lateral sclerosis (ALS), lupusnephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy,Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy andMyasthenia Gravis, without completely shutting down the immune defensecapabilities of complement (i.e., leaving the classical pathway intact).This would leave the C1q-dependent complement activation system intactto handle immune complex processing and to aid in host defense againstinfection.

Compositions for inhibiting LEA-1-mediated complement activation Asdescribed herein, the inventors have unexpectedly discovered thatactivation of LEA-1, leading to lysis, is MASP-3-dependent. As furtherdescribed herein, under physiological conditions, MASP-3-dependent LEA-1activation also contributes to opsonization, thereby providing anadditive effect with LEA-2-mediated complement activation. Asdemonstrated herein, in the presence of Ca⁺⁺, factor D is not required,as MASP-3 can drive activation of LEA-1 in factor D^(−/−) sera. MASP-3,MASP-1, and HTRA-1 are able to convert pro-factor D to active factor D.Likewise, MASP-3 activation appears, in many instances, to be dependenton MASP-1, since MASP-3 (in contrast to MASP-1 and MASP-2) is not anauto-activating enzyme and is incapable of converting into its activeform without the help of MASP-1 (Zundel, S. et al., J. Immunol. 172:4342-4350 (2004); Megyeri et al., J. Biol. Chem. 288:8922-8934 (2013).As MASP-3 does not autoactivate and, in many instances, requires theactivity of MASP-1 to be converted into its enzymatically active form,the MASP-3-mediated activation of the alternative pathway C3 convertaseC3Bb can either be inhibited by targeting the MASP-3 zymogen oralready-activated MASP-3, or by targeting MASP-1-mediated activation ofMASP-3, or both, since, in many instances, in the absence of MASP-1functional activity, MASP-3 remains in its zymogen form and is notcapable of driving LEA-1 through direct formation of the alternativepathway C3 convertase (C3bBb).

Therefore, in one aspect of the invention, the preferred proteincomponent to target in the development of therapeutic agents tospecifically inhibit LEA-1 is an inhibitor of MASP-3 (includinginhibitors of MASP-1-mediated MASP-3 activation (e.g., a MASP-1inhibitor that inhibits MASP-3 activation)).

In accordance with the foregoing, in one aspect, the invention providesmethods of inhibiting the adverse effects of LEA-1 (i.e., hemolysis andopsonization) by administering a MASP-3 inhibitory agent, such as aMASP-3 inhibitory antibody in a subject suffering from, or at risk fordeveloping, a disease or disorder selected from the group consisting ofparoxysmal nocturnal hemoglobinuria (PNH), age-related maculardegeneration (AMD), ischemia-reperfusion injury, arthritis, disseminatedintravascular coagulation, thrombotic microangiopathy (includinghemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome(aHUS) and thrombotic thrombocytopenic purpura (TTP), asthma, densedeposit disease, pauci-immune necrotizing crescentic glomerulonephritis,traumatic brain injury, aspiration pneumonia, endophthalmitis,neuromyelitis optica Behcet's disease, multiple sclerosis, GuillainBarre Syndrome, Alzheimer's disease, Amylotrophic lateral sclerosis(ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabeticretinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy andMyasthenia Gravis, comprising administering to the subject apharmaceutical composition comprising an amount of a MASP-3 inhibitoryagent effective to inhibit MASP-3-dependent complement activation and apharmaceutically acceptable carrier.

MASP-3 inhibitory agents are administered in an amount effective toinhibit MASP-3-dependent complement activation in a living subjectsuffering from, or at risk for developing, paroxysmal nocturnalhemoglobinuria (PNH), age-related macular degeneration (AMD),ischemia-reperfusion injury, arthritis, disseminated intravascularcoagulation, thrombotic microangiopathy (including hemolytic uremicsyndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thromboticthrombocytopenic purpura (TTP)), asthma, dense deposit disease,pauci-immune necrotizing crescentic glomerulonephritis, traumatic braininjury, aspiration pneumonia, endophthalmitis, neuromyelitis optica,Behcet's disease, multiple sclerosis, Guillain Barre Syndrome,Alzheimer's disease, Amylotrophic lateral sclerosis (ALS), lupusnephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy,Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy andMyasthenia Gravis. In the practice of this aspect of the invention,representative MASP-3 inhibitory agents include: molecules that inhibitthe biological activity of MASP-3, including molecules that inhibit atleast one or more of the following: lectin MASP-3-dependent activationof factor B, lectin MASP-3-dependent activation of pro-factor D,MASP-3-dependent, lectin-independent activation of factor B, andMASP-3-dependent, lectin-independent activation of pro-factor D (such assmall-molecule inhibitors, MASP-3 antibodies and fragments thereof, orblocking peptides which interact with MASP-3 or interfere with aprotein-protein interaction), and molecules that decrease the expressionof MASP-3 (such as MASP-3 antisense nucleic acid molecules, MASP-3specific RNAi molecules and MASP-3 ribozymes). A MASP-3 inhibitory agentmay effectively block MASP-3 protein-to-protein interactions, interferewith MASP-3 dimerization or assembly, block Ca⁺⁺ binding, interfere withthe MASP-3 serine protease active site, or reduce MASP-3 proteinexpression, thereby preventing MASP-3 from activating LEA-1-mediated, orlectin-independent, complement activation. The MASP-3 inhibitory agentscan be used alone as a primary therapy or in combination with othertherapeutics as an adjuvant therapy to enhance the therapeutic benefitsof other medical treatments, as further described herein.

High Affinity Monoclonal MASP-3 Inhibitory Antibodies

As described in Examples 11-21 herein, and summarized in TABLES 2A, 2Band TABLE 3 below, the inventors have generated surprisingly highaffinity (i.e. ≤500 pM) MASP-3 inhibitory antibodies that bind to anepitope in the serine protease domain of human MASP-3. As describedherein, the inventors have demonstrated that these high affinity MASP-3antibodies are capable of inhibiting alternative pathway complementactivation in human serum, rodents and non-human primates. The variablelight and heavy chain regions of these antibodies have been sequenced,isolated and analyzed in both a Fab format and in a full-length IgGformat. As described in Example 15 and shown in dendrograms depicted inFIGS. 50A and 50B, the antibodies can be grouped according to sequencesimilarity. A summary of the heavy chain variable regions and the lightchain variable regions of these antibodies is shown in FIGS. 49A and 49Band provided in TABLES 2A and 2B below. Humanized versions ofrepresentative high affinity MASP-3 inhibitory antibodies were generatedas described in Example 19 and are summarized in TABLE 3.

TABLE 2A MASP-3 high affinity inhibitory Antibody Sequences: mouseparental Heavy Light Chain Chain Heavy Light MASP-3 Variable Variablechain chain Antibody Region Region variable variable Reference (amino(amino region region No Group acid) acid) (DNA) (DNA) 4D5 IA SIN: 24SIN: 40 SIN: 217 SIN: 233 1F3 IA SIN: 25 SIN: 41 SIN: 218 SIN: 234 4B6IA SIN: 26 SIN: 42 SIN: 219 SIN: 235 1A10 IA SIN: 27 SIN: 42 SIN: 220SIN: 235 10D12 IB SIN: 28 SIN: 43 SIN: 221 SIN: 236 35C1 IB SIN: 29 SIN:44 SIN: 222 SIN: 237 13B1 IC SIN: 30 SIN: 45 SIN: 223 SIN: 238 1G4 IISIN: 31 SIN: 46 SIN: 224 SIN: 239 1E7 IIIA SIN: 32 SIN: 47 SIN: 225 SIN:240 2D7 IIIA SIN: 33 SIN: 48 SIN: 226 SIN: 241 49C11 IIIA SIN: 34 SIN:49 SIN: 227 SIN: 242 15D9 IIIB SIN: 35 SIN: 50 SIN: 228 SIN: 243 2F5IIIB SIN: 36 SIN: 51 SIN: 229 SIN: 244 1B11 IIIC SIN: 37 SIN: 52 SIN:230 SIN: 245 2F2 IIID SIN: 38 SIN: 53 SIN: 231 SIN: 246 11B6 IIID SIN:39 SIN: 54 SIN: 232 SIN: 247 Note: “SIN” refers to “SEQ ID NO:”

TABLE 2B MASP-3 high affinity inhibitory antibodies: CDRs Heavy LightHeavy Light Chain: Chain: Chain Chain CDR1; CDR1; MASP-3 VariableVariable CDR2; CDR2; Antibody Region Region CDR3 CDR3 Reference (amino(amino (SEQ ID (SEQ ID No. acid) acid) NOs) NOs) 4D5 SIN: 24 SIN: 40 56;58; 60 142; 144; 146 1F3 SIN: 25 SIN: 41 62; 63; 65 149; 144; 146 4B6SIN: 26 SIN: 42 62; 67; 65 149; 144; 146 1A10 SIN: 27 SIN: 42 62; 69; 65149; 144; 146 10D12 SIN: 28 SIN: 43 72; 74; 76 153; 155; 157 35C1 SIN:29 SIN: 44 79; 74; 82 159; 155; 160 13B1 SIN: 30 SIN: 45 84; 86; 88 142;144; 161 1G4 SIN: 31 SIN: 46 91; 93; 95 163; 165; 167 1E7 SIN: 32 SIN:47 109; 110; 112 182; 184; 186 2D7 SIN: 33 SIN: 48 125; 127; 129 196;198; 200 49C11 SIN: 34 SIN: 49 132; 133; 135 203; 165; 204 15D9 SIN: 35SIN: 50 137; 138; 140 206; 207; 208 2F5 SIN: 36 SIN: 51 98; 99; 101 169;171; 173 1B11 SIN: 37 SIN: 52 103; 105; 107 176; 178; 180 2F2 SIN: 38SIN: 53 114; 116; 118 188; 178; 190 11B6 SIN: 39 SIN: 54 114; 121; 123191; 178; 193

TABLE 3 Representative high affinity MASP-3 inhibitory antibodies:humanized and modified to remove post-translational modification sitesHeavy Light Heavy Light Chain: Chain: Chain Chain CDR1; CDR1; MASP-3Variable Variable CDR2; CDR2; Antibody Region aa Region aa CDR3 CDR3Reference (SEQ ID (SEQ ID (SEQ ID (SEQ ID No. NO) NO) NOs) NOs) 4D5parent 24 40 56; 58; 60 142; 144; 146 h4D5-14-1 248 250 56; 58; 60 142;144; 146 h4D5-11-1 249 250 56; 58; 60 142; 144; 146 h4D5-14-1-NA 248 27856; 58; 60 258; 144; 146 h4D5-19-1-NA 249 278 56; 58; 60 258; 144; 14610D12 parent 28 43 72; 74; 76 153; 155; 157 1110D12-45-21 251 253 72;74; 76 153; 155; 157 h10D 12-49-21 252 253 72; 74; 76 153; 155; 157 h10D12-45-21- 251 279 72; 74; 76 263; 155; 157 GA h10D 12-49-21- 252 279 72;74; 76 263; 155; 157 GA 13B1 parent 30 45 84; 86; 88 142; 144; 161h13B1-9-1 254 256 84; 275; 88 142; 144; 161 h13B1-10-1 255 256 84; 86;88 142; 144; 161 h13B1-9-1-NA 254 280 84; 275; 88 258; 144; 161h13B1-10-1-NA 255 280 84; 86; 88 258; 144; 161

Accordingly, in one aspect, the present invention provides an isolatedmonoclonal antibody or antigen-binding fragment thereof thatspecifically binds to the serine protease domain of human MASP-3 (aminoacid residues 450 to 728 of SEQ ID NO:2) with high affinity (having aK_(D) of less than 500 pM), wherein the antibody or antigen-bindingfragment thereof inhibits alternative pathway complement activation. Insome embodiments, the high affinity MASP-3 inhibitory antibody, orantigen-binding fragment thereof inhibits the alternative pathway at amolar ratio of from about 1:1 to about 2.5:1 target MASP-3 to mAb in amammalian subject.

The inhibition of alternative pathway complement activation ischaracterized by at least one or more of the following changes in acomponent of the complement system that occurs as a result ofadministration of a high affinity MASP-3 inhibitory antibody inaccordance with various embodiments of the invention: inhibition ofhemolysis and/or opsonization; inhibition of lectin-independentconversion of factor B; inhibition of lectin-independent conversion offactor D, inhibition of MASP-3 serine protease substrate-specificcleavage; the reduction of hemolysis or the reduction of C3 cleavage andC3b surface deposition; the reduction of Factor B and Bb deposition onan activating surface; the reduction of resting levels (in circulation,and without the experimental addition of an activating surface) ofactive Factor D relative to pro-Factor D; the reduction of levels ofactive Factor D relative to pro-Factor D in response to an activatingsurface; and/or the production of resting and surface-induced levels offluid-phase Ba, Bb, C3b, or C3a.

For example, as described herein the high affinity MASP-3 inhibitoryantibodies, are antibodies or antigen-binding fragments thereof capableof inhibiting factor D maturation (i.e., cleavage of pro-factor D tofactor D) in a mammalian subject. In some embodiments, the high affinityMASP-3 inhibitory antibodies are capable of inhibiting factor Dmaturation in full serum to a level less than 50% than that found inuntreated control serum (such as less than 40%, for example less than30%, such as less than 25%, for example less than 20%, such as less than15%, for example less than 10%, such as less than 5% untreated controlserum not contacted with a MASP-3 inhibitory antibody).

In preferred embodiments, the high affinity MASP-3 inhibitory antibodiesselectively inhibit the alternative pathway, leaving the C1q-dependentcomplement activation system functionally intact.

In another aspect, the present disclosure features a nucleic acidmolecule that encodes one or both of the heavy and light chainpolypeptides of any of the MASP-3 inhibitory antibodies orantigen-binding fragments disclosed herein. Also featured is a vector(e.g., a cloning or expression vector) comprising the nucleic acid and acell (e.g., an insect cell, bacterial cell, fungal cell, or mammaliancell) comprising the vector. The disclosure futher provides a method forproducing any of the MASP-3 inhibitory antibodies or antigen-bindingfragments disclosed herein. The methods include, providing a cellcontaining an expression vector which contains a nucleic acid thatencodes one or both of the heavy and light chain polypeptides of any ofthe antibodies or antigen-binding fragments disclosed herein. The cellor culture of cells is cultured under conditions and for a timesufficient to allow expression by the cell (or culture of cells) of theantibody or antigen-binding fragment thereof encoded by the nucleicacid. The method can also include isolating the antibody or antigenbinding fragment thereof from the cell (or culture of cells) or from themedia in which the cell or cells were cultured.

MASP-3 Epitopes and Peptides

As described in Example 18, illustrated in FIG. 62 and summarized inTABLE 4 below, the high affinity MASP-3 inhibitory antibodies andantigen-binding fragments thereof according to the present inventionwere found to specifically recognize one or more epitopes within theserine protease domain of human MASP-3 (amino acid residues 450 to 728of SEQ ID NO:2). “Specifically recognises” means that the antibody bindsto said epitope with significantly higher affinity than to any othermolecule or part thereof.

TABLE 4 Representative High Affinity MASP-3 inhibitory antibodies: Epitope Binding   Regions of MASP-3 (see also FIG. 62)Peptide Binding Fragments  (Epitopes) with reference to human MASP-3 (w/leader) MASP-3 mAb Ref No. ₄₉₈VLRSQRRDTTVI₅₀₉(SIN: 9)1F3, 4B6, 4D5,  1A10, 10D12, ₄₉₄TAAHVLRSQRRDTTV₅₀₈ 13B1 (SIN: 10)₅₄₄DFNIQNYNHDIALVQ₅₅₈ 1F3, 4B6, 4D5, 1A10 (SIN: 11) ₆₂₆PHAECKTSYESRS₆₃₈13B1 (SIN: 12) ₆₃₉GNYSVTENMFC₆₄₉ 1F3, 4B6, 4D5, 1A10 (SIN: 13)₇₀₄VSNYVDWVWE₇₃₃(SIN: 14) 1F3, 4B6, 4D5, 1A10 ₄₉₈VLRSQRRDTTV₅₀₈(SIN: 15)1F3, 4B6, 4D5, 1A10,  Core sequence of Group I 10D12, 13B1₄₃₅ECGQPSRSLPSLV₄₄₇ 1B11 (SIN: 16) ₄₅₄RNAEPGLFPWQ₄₆₄1G4, 1E7,2D7, 15D9,  (SIN: 17)Core sequence   2F5, 1B11of Groups II and III ₄₇₉KWFGSGALLSASWIL₄₉₃ 15D9, 2F5 (SIN 18)₅₁₄EHVTVYLGLH₅₂₃(SIN: 19) 1E7, 2D7, 1G4 ₅₆₂PVPLGPHVMP₅₇₁(SIN: 20)15D9, 2F5 ₅₈₃APHMLGL₅₈₉(SIN: 21) 1B11 ₆₁₄SDVLQYVKLP₆₂₃(SIN: 22) 1B11₆₆₇AFVIFDDLSQRW₆₇₈(SIN: 23) 1G4, 1E7,2D7, 15D9, 2F5

Accordingly, in some embodiments, the high affinity MASP-3 inhibitoryantibody or antigen-binding fragment thereof specifically binds to anepitope located within the serine protease domain of human MASP-3,wherein said epitope is located within at least one or more of:VLRSQRRDTTVI (SEQ ID NO:9), TAAHVLRSQRRDTTV (SEQ ID NO:10),DFNIQNYNHDIALVQ (SEQ ID NO:11), PHAECKTSYESRS (SEQ ID NO:12),GNYSVTENMFC (SEQ ID NO:13), VSNYVDWVWE (SEQ ID NO:14) and/or VLRSQRRDTTV(SEQ ID NO:15). In some embodiments, the antibody or antigen-bindingfragment thereof binds to an epitope within SEQ ID NO:15. In someembodiments, the antibody or antigen-binding fragment binds to anepitope within SEQ ID NO:9. In some embodiments, the antibody orantigen-binding fragment thereof binds to an epitope within SEQ IDNO:10. In some embodiments, the antibody or antigen-binding fragmentthereof binds to an epitope within SEQ ID NO:12. In some embodidments,the antibody or antigen-binding fragment thereof binds to an epitopewithin SEQ ID NO:10 and SEQ ID NO:12. In some embodiments, the antibodyor antigen-binding fragment thereof binds to an epitope within at leastone of SEQ ID NO:11, SEQ ID NO: 13 and/or SEQ ID NO:14.

In other embodiments, the high affinity MASP-3 inhibitory antibody orantigen-binding fragment thereof specifically binds to an epitopelocated within the serine protease domain of human MASP-3, wherein saidepitope is located within at least one or more of: ECGQPSRSLPSLV (SEQ IDNO:16), RNAEPGLFPWQ (SEQ ID NO:17); KWFGSGALLSASWIL(SEQ ID NO:18);EHVTVYLGLH (SEQ ID NO:19); PVPLGPHVMP (SEQ ID NO:20); APHMLGL (SEQ IDNO:21); SDVLQYVKLP (SEQ ID NO:22); and/or AFVIFDDLSQRW (SEQ ID NO:23).In one embodiment, the antibody or antigen-binding fragment binds to anepitope within SEQ ID NO:17. In one embodiment, the antibody or antigenbinding fragment binds to an epitope within EHVTVYLGLH (SEQ ID NO:19)and/or AFVIFDDLSQRW (SEQ ID NO:23). In one embodiment, the antibody orantigen-binding fragment binds to an epitope within SEQ ID NO:18, SEQ IDNO:20 and/or SEQ ID NO:23. In one embodiment, the antibody orantigen-binding fragment binds to an epitope within at least one of SEQID NO:16, SEQ ID NO: 21 and/or SEQ ID NO:22.

CDR Regions:

In one aspect of the present invention the antibody or functionalequivalent thereof comprises specific hypervariable regions, designatedCDRs. Preferably, the CDRs are CDRs according to the Kabat CDRdefinition. CDRs or hypervariable regions may for example be identifiedby sequence alignment to other antibodies. The CDR regions of the highaffinity MASP-3 inhibitory antibodies are shown in TABLES 18-23.

Group IA mAbs

In one aspect, the invention provides an isolated antibody, orantigen-binding fragment thereof, that binds to MASP-3 comprising: (a) aheavy chain variable region comprising a HC-CDR1 set forth as SEQ IDNO:209 (XXDIN, wherein X at position 1 is S or T and wherein X atposition 2 is N or D); a HC-CDR2 set forth as SEQ ID NO:210(WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D; X at position 8is S, T or R; X at position 9 is I or T; X at position 13 is E or D; Xat position 14 is K or E; and X at position 16 is T or K); and a HC-CDR3set forth as SEQ ID NO:211 (XEDXY, wherein X at position 1 is L or V,and wherein X at position 4 is T or S); and (b) a light chain variableregion comprising a LC-CDR1 set forth as SEQ ID NO:212(KSSQSLLXXRTRKNYLX, wherein X at position 8 is N, I, Q or A; wherein Xat position 9 is S or T; and wherein X at position 17 is A or S); aLC-CDR2 set forth as SEQ ID NO:144 (WASTRES) and a LC-CDR3 set forth asSEQ ID NO:146 (KQSYNLYT). In one embodiment, the HC-CDR1 of the heavychain variable region according to (a) comprises SEQ ID NO:56 (TDDIN).In one embodiment, the HC-CDR1 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:62 (SNDIN). In one embodiment, theHC-CDR2 of the heavy chain variable region according to (a) comprisesSEQ ID NO:58 (WIYPRDDRTKYNDKFK_(D)). In one embodiment, the HC-CDR2 ofthe heavy chain variable region according to (a) comprises SEQ ID NO:63(WIYPRDGSIKYNEKFTD). In one embodiment, the HC-CDR2 of the heavy chainvariable region according to (a) comprises SEQ ID NO:67(WIYPRDGTTKYNEEFTD). In one embodiment, the HC-CDR2 of the heavy chainvariable region according to (a) comprises SEQ ID NO:69(WIYPRDGTTKYNEKFTD). In one embodiment, the HC-CDR3 of the heavy chainvariable region according to (a) comprises SEQ ID NO:60 (LEDTY). In oneembodiment, the HC-CDR3 of the heavy chain variable region according to(a) comprises SEQ ID NO:65 (VEDSY). In one embodiment, the LC-CDR1 ofthe light chain variable region comprises SEQ ID NO:142(KSSQSLLNSRTRKNYLA); SEQ ID NO:257 (KSSQSLLRTRKNYLA), SEQ ID NO:258(KSSQSLLASRTRKNYLA); or SEQ ID NO:259 (KSSQSLLNTRTRKNYLA). In oneembodiment, the LC-CDR1 comprises SEQ ID NO:258 (KSSQSLLASRTRKNYLA). Inone embodiment, the LC-CDR1 comprises SEQ ID NO:149 (KSSQSLLISRTRKNYLS).

In one embodiment, the HC-CDR1 comprises SEQ ID NO:56, the HC-CDR2comprises SEQ ID NO:58, the HC-CDR3 comprises SEQ ID NO:60 and theLC-CDR1 comprises SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQ IDNO:259; the LC-CDR2 comprises SEQ ID NO:144 and the LC-CDR3 comprisesSEQ ID NO:146.

In one embodiment, the HC-CDR1 comprises SEQ ID NO:62, the HC-CDR2comprises SEQ ID NO:63, SEQ ID NO:67 or SEQ ID NO:69, the HC-CDR3comprises SEQ ID NO:65 and the LC-CDR1 comprises SEQ ID NO:149, theLC-CDR2 comprises SEQ ID NO:144 and the LC-CDR3 comprises SEQ ID NO:146.

Group IB mAbs

In another aspect, the invention provides an isolated antibody, orantigen-binding fragment thereof, that binds to MASP-3 comprising: (a) aheavy chain variable region comprising a HC-CDR1 set forth as SEQ IDNO:213 (SYGXX, wherein X at position 4 is M or I and wherein X atposition 5 is S or T); a HC-CDR2 set forth as SEQ ID NO:74; and aHC-CDR3 set forth as SEQ ID NO:214 (GGXAXDY, wherein X at position 3 isE or D and wherein X at position 5 is M or L); and (b) a light chainvariable region comprising a LC-CDR1 set forth as SEQ ID NO:215(KSSQSLLDSXXKTYLX , wherein X at position 10 is D, E or A; wherein X atposition 11 is G or A; and wherein X at position 16 is N or S); aLC-CDR2 set forth as SEQ ID NO:155; and a LC-CDR3 set forth as SEQ IDNO:216 (WQGTHFPXT, wherein X at position 8 is W or Y).

In one embodiment, the HC-CDR1 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:72 (SYGMS). In one embodiment, theHC-CDR1 comprises SEQ ID NO:79 (SYGIT). In one embodiment, the HC-CDR3comprises SEQ ID NO:76 (GGEAMDY). In one embodiment, the HC-CDR3comprises SEQ ID NO:82 (GGDALDY). In one embodiment, the LC-CDR1comprises SEQ ID NO:153 (KSSQSLLDSDGKTYLN); SEQ ID NO:261(KSSQSLLDSEGKTYLN), SEQ ID NO:262 (KSSQSLLDSAGKTYLN) or SEQ ID NO:263(KSSQSLLDSDAKTYLN). In one embodiment, the LC-CDR1 comprises SEQ IDNO:263 (KSSQSLLDSDAKTYLN). In one embodiment, the LC-CDR1 comprises SEQID NO:152. In one embodiment, the LC-CDR3 comprises SEQ ID NO:159(KSSQSLLDSDGKTYLS).

In one embodiment, the LC-CDR3 comprises SEQ ID NO:160 (WQGTHFPYT). Inone embodiment, the HC-CDR1 comprises SEQ ID NO:72, the HC-CDR2comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:76, and theLC-CDR1 comprises SEQ ID NO:153, SEQ ID NO:261, SEQ ID NO:262 or SEQ IDNO:263; the LC-CDR2 comprises SEQ ID NO:155 and the LC-CDR3 comprisesSEQ ID NO:157.

In one embodiment, the HC-CDR comprises SEQ ID NO:72, the HC-CDR2comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:76, and theLC-CDR1 comprises SEQ ID NO:153 or SEQ ID NO:263, the LC-CDR2 comprisesSEQ ID NO:155, and the LC-CDR3 comprises SEQ ID NO:157.

In one embodiment, the HC-CDR1 comprises SEQ ID NO:79, the HC-CDR2comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:82, and theLC-CDR1 comprises SEQ ID NO:159, the LC-CDR2 comprises SEQ ID NO:155 andthe LC-CDR3 comprises SEQ ID NO:160.

Group IC mAbs

In one aspect, the present invention provides an isolated antibody, orantigen-binding fragment thereof, that binds to MASP-3 comprising (a) aheavy chain variable region comprising a HC-CDR1 set forth as SEQ IDNO:84 (GKWIE); a HC-CDR2 set forth as SEQ ID NO:86 (EILPGTGSTNYNEKFKG)or SEQ ID NO:275 (EILPGTGSTNYAQKFQG); and a HC-CDR3 set forth as SEQ IDNO:88 (SEDV); and (b) a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:142 (KSSQSLLNSRTRKNYLA), SEQ ID NO:257(KSSQSLLQSRTRKNYLA); SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ ID NO:259(KSSQSLLNTRTRKNYLA), a LC-CDR2 set forth as SEQ ID NO:144 (WASTRES); anda LC-CDR3 set forth as SEQ ID NO:161 (KQSYNIPT). In one embodiment, theLC-CDR1 comprises SEQ ID NO:258.

Group II mAbs

In one aspect, the present invention provides an isolated antibody, orantigen-binding fragment thereof, that binds to MASP-3 comprising: (a) aheavy chain variable region comprising a HC-CDR1 set forth as SEQ IDNO:91 (GYWIE); a HC-CDR2 set forth as SEQ ID NO:93 (EMLPGSGSTHYNEKFKG),and a HC-CDR3 set forth as SEQ ID NO:95 (SIDY); and (b) a light chainvariable region comprising a LC-CDR1 set forth as SEQ ID NO:163(RSSQSLVQSNGNTYLH), a LC-CDR2 set forth as SEQ ID NO:165 (KVSNRFS) and aLC-CDR3 set forth as SEQ ID NO:167 (SQSTHVPPT).

Group III mAbs

In another aspect, the present invention provides an isolated antibody,or antigen-binding fragment thereof, that binds to MASP-3 comprising:(a) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:109 (RVHFAIRDTNYWMQ), a HC-CDR2 set forth as SEQ ID NO:110(AIYPGNGDTSYNQKFKG), a HC-CDR3 set forth as SEQ ID NO:112 (GSHYFDY); anda light chain variable region comprising a LC-CDR1 set forth as SEQ IDNO:182 (RASQSIGTSIH), a LC-CDR2 set forth as SEQ ID NO:184 (YASESIS) anda LC-CDR3 set forth as SEQ ID NO:186 (QQSNSWPYT); or

(b) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:125 (DYYMN), a HC-CDR2 set forth as SEQ ID NO:127(DVNPNNDGTTYNQKFKG), a HC-CDR3 set forth as SEQ ID NO:129(CPFYYLGKGTHFDY); and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:196 (RASQDISNFLN), a LC-CDR2 set forth as SEQ IDNO:198 (YTSRLHS) and a LC-CDR3 set forth as SEQ ID NO:200 (QQGFTLPWT);or

(c) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:137 a HC-CDR2 set forth as SEQ ID NO:138, a HC-CDR3 set forth asSEQ ID NO:140; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:206, a LC-CDR2 set forth as SEQ ID NO:207 and aLC-CDR3 set forth as SEQ ID NO:208; or

(d) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:98, a HC-CDR2 set forth as SEQ ID NO:99, a HC-CDR3 set forth asSEQ ID NO:101; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:169, a LC-CDR2 set forth as SEQ ID NO:171 and aLC-CDR3 set forth as SEQ ID NO:173; or

(e) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:103, a HC-CDR2 set forth as SEQ ID NO:105, a HC-CDR3 set forth asSEQ ID NO:107; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:176, a LC-CDR2 set forth as SEQ ID NO:178 and aLC-CDR3 set forth as SEQ ID NO:193; or

(f) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:114, a HC-CDR2 set forth as SEQ ID NO:116, a HC-CDR3 set forth asSEQ ID NO:118; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:188, a LC-CDR2 set forth as SEQ ID NO:178 and aLC-CDR3 set forth as SEQ ID NO:190; or

(g) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:114, a HC-CDR2 set forth as SEQ ID NO:121, a HC-CDR3 set forth asSEQ ID NO:123; and a light chain variable region comprising a LC-CDR1set forth as SEQ ID NO:191, a LC-CDR2 set forth as SEQ ID NO:178 and aLC-CDR3 set forth as SEQ ID NO:193; or

(h) a heavy chain variable region comprising a HC-CDR1 set forth as SEQID NO:132, a HC-CDR2 set forth as SEQ ID NO:133, a HC-CDR3 set forth asSEQ ID NO:135; and a light chain variable region comprsing a LC-CDR1 setforth as SEQ ID NO:203, a LC-CDR2 set forth as SEQ ID NO:165 and aLC-CDR3 set forth as SEQ ID NO:204.

Heavy Chain and Light Chain Variable Regions

In one embodiment, the invention provides a high affinity MASP-3inhibitory antibody comprising a heavy chain variable region comprisingor consisting of a sequence which is at least 80%, 85%, 90%, 95%, 98%,99% identical to any of SEQ ID NO:s 24-39, 248-249, 251-252, 254-255 orwherein the antibody comprises a heavy chain variable region comprisingSEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,SEQ ID NO:39, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:251, SEQ IDNO:252, SEQ ID NO:254 or SEQ ID NO:255.

In one embodiment, the invention provides a high affinity MASP-3inhibitory antibody comprising a light chain variable region comprisingor consisting of a sequence which is at least 780%, 85%, 90%, 95%, 98%,99% identical to any of SEQ ID NO:s 40-54, 250, 253, 256, 278, 279, or280 or wherein the antibody comprises a light chain variable regioncomprising SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:250, SEQ ID NO:253, SEQ ID NO:256, SEQ ID NO:278, SEQID NO:279 or SEQ ID NO:280.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:24, SEQ ID NO:248 or SEQ ID NO:249 and a light chaincomprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical toSEQ ID NO:40, SEQ ID NO:250 or SEQ ID NO:278.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:25 and a5 light chain comprising at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to SEQ ID NO:41.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:26 and a light chain comprising at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to SEQ ID NO:42.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:27 and a light chain comprising at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to SEQ ID NO:42.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:28, SEQ ID NO:251 or SEQ ID NO:252 and a light chaincomprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical toSEQ ID NO:43, SEQ ID NO:253 or SEQ ID NO:279.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:29 and a light chain comprising at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to SEQ ID NO:44.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:30, SEQ ID NO:254 or SEQ ID NO:255 and a light chaincomprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical toSEQ ID NO:45, SEQ ID NO:256 or SEQ ID NO:280.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100° Aidentical to SEQ ID NO:31 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100° A identical to SEQ ID NO:46.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100° Aidentical to SEQ ID NO:32 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100° A identical to SEQ ID NO:47.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100° Aidentical to SEQ ID NO:33 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100° A identical to SEQ ID NO:48.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100° Aidentical to SEQ ID NO:34 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100° A identical to SEQ ID NO:49.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100° Aidentical to SEQ ID NO:35 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100° A identical to SEQ ID NO:50.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100° Aidentical to SEQ ID NO:36 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100° A identical to SEQ ID NO:51.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100° Aidentical to SEQ ID NO:37 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100° A identical to SEQ ID NO:52.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100° Aidentical to SEQ ID NO:38 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:53.

In one embodiment, the MASP-3 monoclonal antibody comprises a heavychain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:39 and a light chain comprising at least 80%, 85%, 90%,95%, 98%, 99% or 100% identical to SEQ ID NO:54.

Cross-Competition of High Affinity MASP-3 Antibodies

As described herein, the high affinity MASP-3 inhibitory antibodiesdisclosed herein recognize overlapping epitopes within the serineprotease domain of MASP-3. As described in Example 18, shown in FIGS.61A-E and 62-67, and summarized in TABLES 4 and 28, cross-competitionanalysis and pepscan binding analysis shows that the high affinityMASP-3 inhibitory antibodies cross-compete and bind to common epitopeslocated within the MASP-3 serine protease domain. Thus, in oneembodiment, the invention provides high affinitiy MASP-3 inhibitoryantibodies that specifically recognize an epitope or part thereof withinthe serine protease domain of human MASP-3 recognised by one or moreselected from the group consisting of:

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:24 and a light chain variable region set forth as SEQ IDNO:40;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:25 and a light chain variable region set forth as SEQ IDNO:41;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:26 and a light chain variable region set forth as SEQ IDNO:42;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:27 and a light chain variable region set forth as SEQ IDNO:42;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:28 and a light chain variable region set forth as SEQ IDNO:43;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:29 and a light chain variable region set forth as SEQ IDNO:44;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:30 and a light chain variable region set forth as SEQ IDNO:45;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:31 and a light chain variable region set forth as SEQ IDNO:46;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:32 and a light chain variable region set forth as SEQ IDNO:47;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:33 and a light chain variable region set forth as SEQ IDNO:48;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:34 and a light chain variable region set forth as SEQ IDNO:49;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:35 and a light chain variable region set forth as SEQ IDNO:50;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:36 and a light chain variable region set forth as SEQ IDNO:51;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:37 and a light chain variable region set forth as SEQ IDNO:52;

a monoclonal antibody comprising a heavy chain variable region set forthas SEQ ID NO:38 and a light chain variable region set forth as SEQ IDNO:53; and a monoclonal antibody comprising a heavy chain variableregion set forth as SEQ ID NO:39 and a light chain variable region setforth as SEQ ID NO:54.

According to the present invention, when a given antibody recognises atleast part of an epitope recognised by another given antibody, these twoantibodies are said to recognise the same or overlapping epitopes.

Different assays available to the person skilled in the art may be usedto determine whether an antibody (also designated test antibody)recognises the same or an overlapping epitope as a particular monoclonalantibody (also designated reference antibody). Preferably, the assayinvolves the steps of:

-   -   Providing MASP-3 or a fragment thereof comprising the epitope        recognised by the reference antibody    -   Add the test antibody and the reference antibody to the said        MASP-3, wherein either the test antibody or the reference        antibody is labelled with a detectable label. Alternatively,        both antibodies may be labeled with different detectable labels    -   Detecting the presence of the detectable label at MASP-3    -   Thereby detecting whether the test antibody may displace the        reference antibody

If the reference antibody is displaced, the test antibody recognises thesame or an overlapping epitope as the reference antibody. Thus, if thereference antibody is labeled with a detectable label, then a lowdetectable signal at MASP-3 is indicative of displacement of thereference antibody. If the test antibody is labelled with a detectablelabel, then a high detectable signal at MASP-3 is indicative ofdisplacement of the reference antibody. The MASP-3 fragment maypreferably be immobilised on a solid support enabling facile handling.The detectable label may be any directly or indirectly detectable label,such as an enzyme, a radioactive isotope, a heavy metal, a colouredcompound or a fluorescent compound. In Example 18 in the section“Competition Binding Analysis” herein below describes an exemplarymethod of determining whether a test antibody recognises the same or anoverlapping epitope as a reference antibody is described. The personskilled in the art may easily adapt said method to the particularantibodies in question.

The MASP-3 antibodies useful in this aspect of the invention includemonoclonal or recombinant antibodies derived from any antibody producingmammal and may be multispecific (i.e., bispecific or trispecific),chimeric, humanized, fully human, anti-idiotype, and antibody fragments.Antibody fragments include Fab, Fab′, F(ab)₂, F(ab′)₂, Fv fragments,scFv fragments and single-chain antibodies as further described herein.

MASP-3 antibodies can be screened for the ability to inhibit alternativepathway complement activation system using the assays described herein.The inhibition of alternative pathway complement activation ischaracterized by at least oneor more of the following changes in acomponent of the complement system that occurs as a result ofadministration of a high affinity MASP-3 inhibitory antibody inaccordance with various embodiments of the invention: inhibition ofhemolysis and/or opsonization; inhibition of lectin-independentconversion of factor B; inhibition of lectin-independent conversion offactor D, inhibition of MASP-3 serine protease substrate-specificcleavage; the reduction of hemolysis or the reduction of C3 cleavage andC3b surface deposition; the reduction of Factor B and Bb deposition onan activating surface; the reduction of resting levels (in circulation,and without the experimental addition of an activating surface) ofactive Factor D relative to pro-Factor D; the reduction of levels ofactive Factor D relative to pro-Factor D in response to an activatingsurface; and/or the production of resting and surface-induced levels offluid-phase Ba, Bb, C3b, or C3a.

MASP-3 Antibodies with Reduced Effector Function

In some embodiments of this aspect of the invention, the high affinityMASP-3 inhibitory antibodies described herein have reduced effectorfunction in order to reduce inflammation that may arise from theactivation of the classical complement pathway. The ability of IgGmolecules to trigger the classical complement pathway has been shown toreside within the Fc portion of the molecule (Duncan, A. R., et al.,Nature 332:738-740 (1988)). IgG molecules in which the Fc portion of themolecule has been removed by enzymatic cleavage are devoid of thiseffector function (see Harlow, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1988). Accordingly, antibodies withreduced effector function can be generated as the result of lacking theFc portion of the molecule by having a genetically engineered Fcsequence that minimizes effector function, or being of either the humanIgG₂ or IgG₄ isotype.

Antibodies with reduced effector function can be produced by standardmolecular biological manipulation of the Fc portion of the IgG heavychains as described in Jolliffe et al., Int'l Rev. Immunol. 10:241-250,(1993), and Rodrigues et al., J. Immunol. 151:6954-6961, (1998).Antibodies with reduced effector function also include human IgG2 andIgG4 isotypes that have a reduced ability to activate complement and/orinteract with Fc receptors (Ravetch, J. V., et al., Annu. Rev. Immunol.9:457-492, (1991); Isaacs, J. D., et al.,J. Immunol. 148:3062-3071,1992; van de Winkel, J. G., et al., Immunol. Today 14:215-221, (1993)).Humanized or fully human antibodies specific to human MASP-1, MASP-2 orMASP-3 (including dual, pan, bispecific or trispecific antibodies)comprised of IgG2 or IgG4 isotypes can be produced by one of severalmethods known to one of ordinary skilled in the art, as described inVaughan, T. J., et al., Nature Biotechnical 16:535-539, (1998).

Production of High Affinity MASP-3 Inhibitory Antibodies

MASP-3 antibodies can be produced using MASP-3 polypeptides (e.g.,full-length MASP-3) or using antigenic MASP- 3 epitope-bearing peptides(e.g., a portion of the MASP-3 polypeptide), for example as described inExample 14 herein below. Immunogenic peptides may be as small as fiveamino acid residues. The MASP-3 peptides and polypeptides used to raiseantibodies may be isolated as natural polypeptides, or recombinant orsynthetic peptides and catalytically inactive recombinant polypeptides.Antigens useful for producing MASP-3 antibodies also include fusionpolypeptides, such as fusions of a MASP-3 polypeptide or a portionthereof with an immunoglobulin polypeptide or with maltose-bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is hapten-like, such portionmay be advantageously joined or linked to a macromolecular carrier (suchas keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) ortetanus toxoid) for immunization.

Monoclonal Antibodies

As used herein, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogenous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. Monoclonal antibodies can be obtainedusing any technique that provides for the production of antibodymolecules by continuous cell lines in culture, such as the hybridomamethod described by Kohler, G., et al., Nature 256:495, (1975), or theymay be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567 to Cabilly). Monoclonal antibodies may also be isolated fromphage antibody libraries using the techniques described in Clackson, T.,et al., Nature 352:624-628, (1991), and Marks, J. D., et al., J. Mol.Biol. 222:581-597, (1991). Such antibodies can be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

For example, monoclonal antibodies can be obtained by injecting asuitable mammal (e.g., a BALB/c mouse) with a composition comprising aMASP-3 polypeptide, or portion thereof. After a predetermined period oftime, splenocytes are removed from the mouse and suspended in a cellculture medium. The splenocytes are then fused with an immortal cellline to form a hybridoma. The formed hybridomas are grown in cellculture and screened for their ability to produce a monoclonal antibodyagainst MASP-3. (See also Current Protocols in Immunology, Vol. 1., JohnWiley & Sons, pages 2.5.1-2.6.7, 1991.)

Human monoclonal antibodies may be obtained through the use oftransgenic mice that have been engineered to produce specific humanantibodies in response to antigenic challenge. In this technique,elements of the human immunoglobulin heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous immunoglobulin heavychain and light chain loci. The transgenic mice can synthesize humanantibodies specific for human antigens, such as the MASP-2 antigensdescribed herein, and the mice can be used to produce human MASP-2antibody-secreting hybridomas by fusing B-cells from such animals tosuitable myeloma cell lines using conventional Kohler-Milsteintechnology. Methods for obtaining human antibodies from transgenic miceare described, for example, by Green, L. L., et al., Nature Genet. 7:13,1994; Lonberg, N., et al., Nature 368:856, 1994; and Taylor, L. D., etal., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, The Humana Press, Inc., Vol. 10, pages 79-104, 1992).

Once produced, monoclonal antibodies are first tested for specificMASP-3 binding or, where desired, dual MASP-1/3, MASP-2/3 or MASP-1/2binding. Methods for determining whether an antibody binds to a proteinantigen and/or the affinity for an antibody to a protein antigen areknown in the art. For example, the binding of an antibody to a proteinantigen can be detected and/or quantified using a variety of techniquessuch as, but not limited to, Western blot, dot blot, plasmon surfaceresonance method (e.g., BlAcore system; Pharmacia Biosensor AB, Uppsala,Sweden and Piscataway, N.J.), or enzyme-linked immunosorbent assays(ELISA). See, e.g., Harlow and Lane (1988) “Antibodies: A LaboratoryManual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.;Benny K. C. Lo (2004) “Antibody Engineering: Methods and Protocols,”Humana Press (ISBN: 1588290921); Borrebaek (1992) “Antibody Engineering,A Practical Guide,” W.H. Freeman and Co., NY; Borrebaek (1995) “AntibodyEngineering,” 2^(nd) Edition, Oxford University Press, NY, Oxford; Johneet al. (1993), Immunol. Meth. 160:191-198; Jonsson et al. (1993) Ann.Biol. Clin. 51: 19-26; and Jonsson et al. (1991) Biotechniques11:620-627. See also, U.S. Pat. No. 6,355,245.

The affinity of MASP-3 monoclonal antibodies can be readily determinedby one of ordinary skill in the art (see, e.g., Scatchard, A., NY Acad.Sci. 51:660-672, 1949). In one embodiment, the MASP-3 monoclonalantibodies useful for the methods of the invention bind to MASP-3 with abinding affinity of <100 nM, preferably <10 nM, preferably <2 nM, andmost preferably with high affinity of <500 pM.

Once antibodies are identified that specifically bind to MASP-3, theMASP-3 antibodies are tested for the ability to function as analternative pathway inhibitor in one of several functional assays, suchas, for example, the inhibition of alternative pathway complementactivation is characterized by at least one or more of the followingchanges in a component of the complement system that occurs as a resultof administration of a high affinity MASP-3 inhibitory antibody inaccordance with various embodiments of the invention: inhibition ofhemolysis and/or opsonization; inhibition of lectin-independentconversion of factor B; inhibition of lectin-independent conversion offactor D, inhibition of MASP-3 serine protease substrate-specificcleavage; the reduction of hemolysis or the reduction of C3 cleavage andC3b surface deposition; the reduction of Factor B and Bb deposition onan activating surface; the reduction of resting levels (in circulation,and without the experimental addition of an activating surface) ofactive Factor D relative to pro-Factor D; the reduction of levels ofactive Factor D relative to pro-Factor D in response to an activatingsurface; the reduction in production of resting and surface-inducedlevels of fluid-phase Ba, Bb, C3b, or C3a; and/or the the reduction indeposition of factor P.

Chimeric/Humanized Antibodies

Monoclonal antibodies useful in the method of the invention includechimeric antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies (U.S. Pat. No. 4,816,567, toCabilly; and Morrison, S. L., et al., Proc. Nat'l Acad. Sci. USA81:6851-6855, (1984)).

One form of a chimeric antibody useful in the invention is a humanizedmonoclonal MASP-3 antibody. Humanized forms of non-human (e.g., murine)antibodies are chimeric antibodies, which contain minimal sequencederived from non-human immunoglobulin. Humanized monoclonal antibodiesare produced by transferring the non-human (e.g., mouse) complementaritydetermining regions (CDR), from the heavy and light variable chains ofthe mouse immunoglobulin into a human variable domain. Typically,residues of human antibodies are then substituted in the frameworkregions of the non-human counterparts. Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domainsin which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe Fv framework regions are those of a human immunoglobulin sequence.The humanized antibody optionally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones, P. T., et al., Nature321:522-525, (1986); Reichmann, L., et al., Nature 332:323-329, (1988);and Presta, Curr. Op. Struct. Biol. 2:593-596, (1992).

The humanized antibodies useful in the invention include humanmonoclonal antibodies including at least a MASP-3 binding CDR3 region.In addition, the Fc portions may be replaced so as to produce IgA or IgMas well as human IgG antibodies. Such humanized antibodies will haveparticular clinical utility because they will specifically recognizehuman MASP-3 but will not evoke an immune response in humans against theantibody itself. Consequently, they are better suited for in vivoadministration in humans, especially when repeated or long-termadministration is necessary

Techniques for producing humanized monoclonal antibodies are alsodescribed, for example, by Jones, P. T., et al., Nature 321:522, (1986);Carter, P., et al., Proc. Nat'l. Acad. Sci. USA 89:4285, (1992); Sandhu,J. S., Crit. Rev. Biotech. 12:437, (1992); Singer, I. I., et al., J.Immun. 150:2844, (1993); Sudhir (ed.), Antibody Engineering Protocols,Humana Press, Inc., (1995); Kelley, “Engineering TherapeuticAntibodies,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), John Wiley & Sons, Inc., pages 399-434, (1996); and by U.S.Pat. No. 5,693,762, to Queen, 1997. In addition, there are commercialentities that will synthesize humanized antibodies from specific murineantibody regions, such as Protein Design Labs (Mountain View, Calif.).

Recombinant antibodies

MASP-3 antibodies can also be made using recombinant methods. Forexample, human antibodies can be made using human immunoglobulinexpression libraries (available for example, from Stratagene, Corp., LaJolla, Calif.k) to produce fragments of human antibodies (V_(H), V_(L),Fv, Factor D, Fab or F(ab′)₂). These fragments are then used toconstruct whole human antibodies using techniques similar to those forproducing chimeric antibodies.

Immunoglobulin Fragments

The MASP-3 inhibitory agents useful in the method of the inventionencompass not only intact immunoglobulin molecules but also thewell-known fragments including Fab, Fab′, F(ab)₂, F(ab′)₂ and Fvfragments, scFv fragments, diabodies, linear antibodies, single-chainantibody molecules and multispecific (e.g., bispecific and trispecific)antibodies formed from antibody fragments.

It is well known in the art that only a small portion of an antibodymolecule, the paratope, is involved in the binding of the antibody toits epitope (see, e.g., Clark, W. R., The Experimental Foundations ofModern Immunology, Wiley & Sons, Inc., NY, 1986). The pFc′ and Fcregions of the antibody are effectors of the classical complementpathway but are not involved in antigen binding. An antibody from whichthe pFc′ region has been enzymatically cleaved, or which has beenproduced without the pFc′ region, is designated an F(ab′)₂ fragment andretains both of the antigen binding sites of an intact antibody. Anisolated F(ab′)₂ fragment is referred to as a bivalent monoclonalfragment because of its two antigen binding sites. Similarly, anantibody from which the Fc region has been enzymatically cleaved, orwhich has been produced without the Fc region, is designated a Fabfragment, and retains one of the antigen binding sites of an intactantibody molecule.

Antibody fragments can be obtained by proteolytic hydrolysis, such as bypepsin or papain digestion of whole antibodies by conventional methods.For example, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent to produce3.5S Fab′ monovalent fragments. Optionally, the cleavage reaction can beperformed using a blocking group for the sulfhydryl groups that resultfrom cleavage of disulfide linkages. As an alternative, an enzymaticcleavage using pepsin produces two monovalent Fab fragments and an Fcfragment directly. These methods are described, for example, U.S. Pat.No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem.Biophys. 89:230, (1960); Porter, R. R., Biochem. 1 73:119, (1959);Edelman, et al., in Methods in Enzymology 1:422, Academic Press, (1967);and by Coligan at pages 2.8.1-2.8.10 and 2.10-2.10.4.

In some embodiments, the use of antibody fragments lacking the Fc regionare preferred to avoid activation of the classical complement pathwaywhich is initiated upon binding Fc to the Fcy receptor. There areseveral methods by which one can produce a monoclonal antibody thatavoids Fcy receptor interactions. For example, the Fc region of amonoclonal antibody can be removed chemically using partial digestion byproteolytic enzymes (such as ficin digestion), thereby generating, forexample, antigen-binding antibody fragments such as Fab or F(ab)₂fragments (Mariani, M., et al., Mol. Immunol. 28:69-71, (1991)).Alternatively, the human γ4 IgG isotype, which does not bind Fcyreceptors, can be used during construction of a humanized antibody asdescribed herein. Antibodies, single chain antibodies andantigen-binding domains that lack the Fc domain can also be engineeredusing recombinant techniques described herein.

Single-Chain Antibody Fragments

Alternatively, one can create single peptide chain binding moleculesspecific for MASP-3 in which the heavy and light chain Fv regions areconnected. The Fv fragments may be connected by a peptide linker to forma single-chain antigen binding protein (scFv). These single-chainantigen binding proteins are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains which areconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are described for example, by Whitlow, etal., “Methods: A Companion to Methods in Enzymology” 2:97, (1991); Bird,et al., Science 242:423, (1988); U.S. Pat. No. 4,946,778, to Ladner;Pack, P., et al., Bio/Technology 11:1271, (1993).

As an illustrative example, a MASP-3-specific scFv can be obtained byexposing lymphocytes to MASP-3 polypeptide in vitro and selectingantibody display libraries in phage or similar vectors (for example,through the use of immobilized or labeled MASP-3 protein or peptide).Genes encoding polypeptides having potential MASP-3 polypeptide bindingdomains can be obtained by screening random peptide libraries displayedon phage or on bacteria such as E. coli. These random peptide displaylibraries can be used to screen for peptides which interact with MASP-3.Techniques for creating and screening such random peptide displaylibraries are well known in the art (U.S. Pat. No. 5,223,409, toLardner; U.S. Pat. No. 4,946,778, to Ladner; U.S. Pat. No. 5,403,484, toLardner; U.S. Pat. No. 5,571,698, to Lardner; and Kay et al., PhageDisplay of Peptides and Proteins Academic Press, Inc., 1996) and randompeptide display libraries and kits for screening such libraries areavailable commercially, for instance from CLONTECH Laboratories, Inc.(Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif), New EnglandBiolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc.(Piscataway, N.J.).

Another form of a MASP-3 antibody fragment useful in this aspect of theinvention is a peptide coding for a single complementarity-determiningregion (CDR) that binds to an epitope on a MASP-3 antigen and inhibitsalternative complement pathway activation.

CDR peptides (“minimal recognition units”) can be obtained byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells(see, for example, Larrick et al., Methods: A Companion to Methods inEnzymology 2:106, (1991); Courtenay-Luck, “Genetic Manipulation ofMonoclonal Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), page 166,Cambridge University Press, (1995); and Ward et al., “GeneticManipulation and Expression of Antibodies,” in Monoclonal Antibodies:Principles and Applications, Birch et al. (eds.), page 137, Wiley-Liss,Inc., 1995).

The high affinity MASP-3 inhibitory antibodies described herein areadministered to a subject in need thereof to inhibit alternative pathwayactivation. In some embodiments, the high affinity MASP-3 inhibitoryantibody is a humanized monoclonal MASP-3 antibody. optionally withreduced effector function.

Bispecific Antibodies

The high affinity MASP-3 inhibitory antibodies useful in the method ofthe invention encompass multispecific (i.e., bispecific and trispecific)antibodies. Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In one embodiment, the compositions and methodscomprise the use of a bispecific antibody comprising a bindingspecificity for the serine protease domain of MASP-3 and a bindingspecificity for MASP-2 (e.g., binding to at least one of CCP1-CCP2 orserine protease domain of MASP-2). In another embodiment, the methodcomprises the use of a bispecific antibody comprising a bindingspecificity for the serine protease domain of MASP-3 and a bindingspecificity for MASP-1 (e.g., binding to the serine protease domain ofMASP-1). In another embodiment, the method comprises the use of atrispecific antibody comprising a binding specificity for MASP-3 (e.g.,binding to the serine protease domain of MASP-3), a binding specificityfor MASP-2 (e.g., binding to at least one of CCP1-CCP2 or serineprotease domain of MASP-2) and a binding specificity for MASP-1 (e.g.,binding to the serine protease domain of MASP-1).

Methods for making bispecific antibodies are within the purview of thoseskilled in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy-chain/light-chain pairs, where the two heavy chainshave different specificities (Milstein and Cuello, Nature 305:537-539(1983)). Antibody variable domains with the desired bindingspecificities (antibody-antigen combining sites) can be fused toimmunoglobulin constant domain sequences. The fusion preferably is withan immunoglobulin heavy-chain constant domain, including at least partof the hinge, C_(H)2, and C_(H)3 regions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host organism. For further details ofillustrative currently known methods for generating bispecificantibodies see, e.g., Suresh et al., Methods in Enzymology 121:210(1986); WO96/27011; Brennan et al., Science 229:81 (1985); Shalaby etal., J. Exp. Med. 175:217-225 (1992); Kostelny et al., J. Immunol.148(5):1547-1553 (1992); Hollinger et al. Proc. Natl. Acad. Sci USA90:6444-6448 (1993); Gruber et al., J. Immunol. 152:5368 (1994); andTutt et al., J. Immunol. 147:60 (1991). Bispecific antibodies alsoinclude cross-linked or heteroconjugate antibodies. Heteroconjugateantibodies may be made using any convenient cross -linking methods.Suitable crosslinking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. (See, e.g., Kostelny et al. J. Immunol.148(5):1547-1553 (1992)). The “diabody” technology described byHollinger et al. Proc. Natl. Acad. Sci USA 90:6444-6448 (1993), hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (VH)connected to a light-chain variable domain (VL) by a linker which is tooshort to allow pairing between the two domains on the same chain.Accordingly, the VH and VL domains of one fragment are forced to pairwith the complementary VL and VH domains of another fragment, therebyforming two antigen-binding sites. Bispecific diabodies, as opposed tobispecific whole antibodies, may also be particularly useful becausethey can be readily constructed and expressed in E. coli. Diabodies (andmany other polypeptides such as antibody fragments) of appropriatebinding specificities can be readily selected using phage display(WO94/13804) from libraries. If one arm of the diabody is to be keptconstant, for instance, with a specificity directed against antigen X,then a library can be made where the other arm is varied and an antibodyof appropriate specificity selected.

Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (scFv) dimers has also been reported. (See, e.g., Gruberet al. J. Immunol., 152:5368 (1994)). Alternatively, the antibodies canbe “linear antibodies” as described in, e.g., Zapata et al., ProteinEng. 8(10):1057-1062 (1995). Briefly described, these antibodiescomprise a pair of tandem Factor D segments (V_(H)-C_(H)I-V_(H)-C_(H)I)which form a pair of antigen binding regions. Linear antibodies can bebispecific or monospecific. The methods of the invention also embracethe use of variant forms of bispecific antibodies such as thetetravalent dual variable domain immunoglobulin (DVD-Ig) moleculesdescribed in Wu et al., Nat Biotechnol 25:1290-1297 (2007). The DVD-Igmolecules are designed such that two different light chain variabledomains (VL) from two different parent antibodies are linked in tandemdirectly or via a short linker by recombinant DNA techniques, followedby the light chain constant domain. Methods for generating DVD-Igmolecules from two parent antibodies are further described in, e.g.,WO08/024188 and WO07/024715, the disclosures of each of which areincorporated herein by reference in their entirety.

III. PHARMACEUTICAL COMPOSITIONS AND DELIVERY METHODS DOSING

In another aspect, the invention provides compositions comprising highaffinity MASP-3 inhibitory antibodies for inhibiting the adverse effectsof alternative pathway complement activation in a subject in needthereof, such as, for example, a subject suffering from an alternativepathway-related disease or condition, such as, for example a hemolyticdisease, such as PNH, or a disease or disorder selected from the groupconsisting of age-related macular degeneration (AMD),ischemia-reperfusion injury, arthritis, disseminated intravascularcoagulation, thrombotic microangiopathy (including hemolytic uremicsyndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thromboticthrombocytopenic purpura (TTP)), asthma, dense deposit disease,pauci-immune necrotizing crescentic glomerulonephritis, traumatic braininjury, aspiration pneumonia, endophthalmitis, neuromyelitis optica,Behcet's disease, multiple sclerosis (MS), Guillain Barre Syndrome,Alzheimer's disease, Amylotrophic lateral sclerosis (ALS), lupusnephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy,Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy andMyasthenia Gravis.

The methods of this aspect of the invention comprises administering tothe subject a composition comprising an amount of a high affinity MASP-3inhibitory antibody effective to inhibit alternative pathway complementactivation and a pharmaceutically acceptable carrier. In someembodiments, the method further comprises administering a compositioncomprising a MASP-2 inhibitory agent. The high affinity MASP-3inhibitory antibodies and MASP-2 inhibitory agents can be administeredto a subject in need thereof, at therapeutically effective doses totreat or ameliorate conditions associated with alternative pathwaycomplement activation, and optionally also MASP-2-dependent complementactivation. A therapeutically effective dose refers to the amount of theMASP-3 inhibitory antibody, or a combination of a MASP-3 inhibitoryantibody and a MASP-2 inhibitory agent sufficient to result inamelioration of symptoms of the condition. The inhibition of alternativepathway complement activation is characterized by at least oneor more ofthe following changes in a component of the complement system thatoccurs as a result of administration of a high affinity MASP-3inhibitory antibody in accordance with various embodiments of theinvention: inhibition of hemolysis and/or opsonization; inhibition oflectin-independent conversion of factor B; inhibition oflectin-independent conversion of factor D, inhibition of MASP-3 serineprotease substrate-specific cleavage; the reduction of hemolysis or thereduction of C3 cleavage and C3b surface deposition; the reduction ofFactor B and Bb deposition on an activating surface; the reduction ofresting levels (in circulation, and without the experimental addition ofan activating surface) of active Factor D relative to pro-Factor D; thereduction of levels of active Factor D relative to pro-Factor D inresponse to an activating surface; and/or the the reduction in theproduction of resting and surface-induced levels of fluid-phase Ba, Bb,C3b, or C3a.

Toxicity and therapeutic efficacy of MASP-3 and MASP-2 inhibitory agentscan be determined by standard pharmaceutical procedures employingexperimental animal models. Using such animal models, the NOAEL (noobserved adverse effect level) and the MED (the minimally effectivedose) can be determined using standard methods. The dose ratio betweenNOAEL and MED effects is the therapeutic ratio, which is expressed asthe ratio NOAEL/MED MASP-3 inhibitory agents and MASP-2 inhibitoryagents that exhibit large therapeutic ratios or indices are mostpreferred. The data obtained from the cell culture assays and animalstudies can be used in formulating a range of dosages for use in humans.The dosage of the MASP-3 inhibitory agent and MASP-2 inhibitory agentpreferably lies within a range of circulating concentrations thatinclude the IVIED with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized.

For any compound formulation, the therapeutically effective dose can beestimated using animal models. For example, a dose may be formulated inan animal model to achieve a circulating plasma concentration range thatincludes the MED. Quantitative levels of the MASP-3 inhibitory agent orMASP-2 inhibitory agent in plasma may also be measured, for example, byhigh performance liquid chromatography.

In addition to toxicity studies, effective dosage may also be estimatedbased on the amount of target MASP protein present in a living subjectand the binding affinity of the MASP-3 or MASP-2 inhibitory agent.

It has been reported that MASP-1 levels in normal human subjects ispresent in serum in levels in the range of from 1.48 to 12.83 μg/mL(Terai I. et al, Clin Exp Immunol 110:317-323 (1997); Theil et al.,Clin. Exp. Immunol. 169:38 (2012)). The mean serum MASP-3 concentrationsin normal human subjects has been reported to be in the range of about2.0 to 12.9 μg/mL (Skjoedt M et al., Immunobiology 215(11):921-31(2010); Degn et al., J. Immunol Methods, 361-37 (2010); Csuka et al.,Mol. Immunol. 54:271 (2013). It has been shown that MASP-2 levels innormal human subjects is present in serum in low levels in the range of500 ng/mL, and MASP-2 levels in a particular subject can be determinedusing a quantitative assay for MASP-2 described in Moller-Kristensen M.,et al., J. Immunol. Methods 282:159-167 (2003) and Csuka et al., Mol.Immunol. 54:271 (2013).

Generally, the dosage of administered compositions comprising MASP-3inhibitory agents or MASP-2 inhibitory agents varies depending on suchfactors as the subject's age, weight, height, sex, general medicalcondition, and previous medical history. As an illustration, MASP-3inhibitory agents or MASP-2 inhibitory agents (such as MASP-3antibodies, MASP-1 antibodies or MASP-2 antibodies), can be administeredin dosage ranges from about 0.010 to 100.0 mg/kg, preferably 0.010 to 10mg/kg, preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kgof the subject body weight. In some embodiments, MASP-2 inhibitoryagents (such as MASP-2 antibodies) are administered in dosage rangesfrom about preferably 0.010 to 10 mg/kg, preferably 0.010 to 1.0 mg/kg,more preferably 0.010 to 0.1 mg/kg of the subject body weight. In someembodiments, MASP-1 inhibitory agents (such as MASP-1 antibodies) orMASP-3 inhibitory agents (such as MASP-3 antibodies) are administered indosage ranges from about 0.010 to 100.0 mg/kg, preferably 0.010 to 10mg/kg, such as form about 1 mg/kg to about 10 mg/kg, preferably 0.010 to1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of the subject bodyweight.

Therapeutic efficacy of MASP-3 inhibitory compositions, optionally incombination with MASP-2 inhibitory compositions, or of MASP-1 inhibitorycompositions, optionally in combination with MASP-2 inhibitorycompositions, and methods of the present invention in a given subject,and appropriate dosages, can be determined in accordance with complementassays well known to those of skill in the art. Complement generatesnumerous specific products. During the last decade, sensitive andspecific assays have been developed and are available commercially formost of these activation products, including the small activationfragments C3a, C4a, and C5a and the large activation fragments iC3b,C4d, Bb, and sC5b-9. Most of these assays utilize monoclonal antibodiesthat react with new antigens (neoantigens) exposed on the fragment, butnot on the native proteins from which they are formed, making theseassays very simple and specific. Most rely on ELISA technology, althoughradioimmunoassay is still sometimes used for C3a and C5a. These latterassays measure both the unprocessed fragments and their ‘desArg’fragments, which are the major forms found in the circulation.Unprocessed fragments and CS_(adesArg) are rapidly cleared by binding tocell surface receptors and are hence present in very low concentrations,whereas C3adesAr_(g) does not bind to cells and accumulates in plasma.Measurement of C3a provides a sensitive, pathway-independent indicatorof complement activation. Alternative pathway activation can be assessedby measuring the Bb fragment and/or measurement of factor D activation.Detection of the fluid-phase product of membrane attack pathwayactivation, sC5b-9, provides evidence that complement is being activatedto completion. Because both the lectin and classical pathways generatethe same activation products, C4a and C4d, measurement of these twofragments does not provide any information about which of these twopathways has generated the activation products.

The inhibition of the alternative pathway in a mammalian subject ischaracterized by at least one or more of the following in the mammaliansubject after treatment with a high affinity MASP-3 inhibitory antibodydisclosed herein: inhibition of Factor D maturation; inhibition of thealternative pathway when administered to the subject at a molar ratio offrom about 1:1 to about 2.5:1 (MASP-3 target to mAb); the classicalpathway is not inhibited; inhibition of hemolysis and/or opsonization; areduction of hemolysis or the reduction of C3 cleavage and C3b surfacedeposition; a reduction of Factor B and Bb deposition on an activatingsurface; a reduction of resting levels (in circulation, and without theexperimental addition of an activating surface) of active Factor Drelative to pro-Factor D; a reduction of levels of active Factor Drelative to pro-Factor D in response to an activating surface; and/or areduction of the production of resting and surface-induced levels offluid-phase Ba, Bb, C3b, or C3a.

The inhibition of MASP-2-dependent complement activation ischaracterized by at least one of the following changes in a component ofthe complement system that occurs as a result of administration of aMASP-2 inhibitory agent in accordance with the methods of the invention:the inhibition of the generation or production of MASP-2-dependentcomplement activation system products C4b, C3a, C5a and/or C5b-9 (MAC)(measured, for example, as described in measured, for example, asdescribed in Example 2 of U.S. Pat. No. 7,919,094), the reduction of C4cleavage and C4b deposition or the reduction of C3 cleavage and C3bdeposition.

Pharmaceutical Carriers and Delivery Vehicles

In general, the MASP-3 inhibitory antibody compositions, or compositionscomprising a combination of MASP-2 and MASP-3 inhibitory agents, may becombined with any other selected therapeutic agents, are suitablycontained in a pharmaceutically acceptable carrier. The carrier isnon-toxic, biocompatible and is selected so as not to detrimentallyaffect the biological activity of the MASP-3 inhibitory antibody or theMASP-2 inhibitory agent (and any other therapeutic agents combinedtherewith). Exemplary pharmaceutically acceptable carriers for peptidesare described in U.S. Pat. No. 5,211,657 to Yamada. The MASP-3antibodies useful in the invention, as described herein, may beformulated into preparations in solid, semi-solid, gel, liquid orgaseous forms such as tablets, capsules, powders, granules, ointments,solutions, depositories, inhalants and injections allowing for oral,parenteral or surgical administration. The invention also contemplateslocal administration of the compositions by coating medical devices andthe like.

Suitable carriers for parenteral delivery via injectable, infusion orirrigation and topical delivery include distilled water, physiologicalphosphate-buffered saline, normal or lactated Ringer's solutions,dextrose solution, Hank's solution, or propanediol. In addition,sterile, fixed oils may be employed as a solvent or suspending medium.For this purpose any biocompatible oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables. The carrier and agentmay be compounded as a liquid, suspension, polymerizable ornon-polymerizable gel, paste or salve.

The carrier may also comprise a delivery vehicle to sustain (i.e.,extend, delay or regulate) the delivery of the agent(s) or to enhancethe delivery, uptake, stability or pharmacokinetics of the therapeuticagent(s). Such a delivery vehicle may include, by way of non-limitingexample, microparticles, microspheres, nanospheres or nanoparticlescomposed of proteins, liposomes, carbohydrates, synthetic organiccompounds, inorganic compounds, polymeric or copolymeric hydrogels andpolymeric micelles. Suitable hydrogel and micelle delivery systemsinclude the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexesdisclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrincomplexes disclosed in U.S. Patent Application Publication No.2002/0019369 A1. Such hydrogels may be injected locally at the site ofintended action, or subcutaneously or intramuscularly to form asustained release depot.

Compositions of the present invention may be formulated for deliverysubcutaneously, intra-muscularly, intravenously, intra-arterially or asan inhalant.

For intra-articular delivery, the MASP-3 inhibitory antibody, optionallyin combination with a MASP-2 inhibitory agent may be carried inabove-described liquid or gel carriers that are injectable,above-described sustained-release delivery vehicles that are injectable,or a hyaluronic acid or hyaluronic acid derivative.

For oral administration of non-peptidergic agents, the MASP-3 inhibitoryantibody, optionally in combination with a MASP-2 inhibitory agent maybe carried in an inert filler or diluent such as sucrose, cornstarch, orcellulose.

For topical administration, the MASP-3 inhibitory antibody, optionallyin combination with a MASP-2 inhibitory agent may be carried inointment, lotion, cream, gel, drop, suppository, spray, liquid orpowder, or in gel or microcapsular delivery systems via a transdermalpatch.

Various nasal and pulmonary delivery systems, including aerosols,metered-dose inhalers, dry powder inhalers, and nebulizers, are beingdeveloped and may suitably be adapted for delivery of the presentinvention in an aerosol, inhalant, or nebulized delivery vehicle,respectively.

For intrathecal (IT) or intracerebroventricular (ICV) delivery,appropriately sterile delivery systems (e.g., liquids; gels,suspensions, etc.) can be used to administer the present invention.

The compositions of the present invention may also include biocompatibleexcipients, such as dispersing or wetting agents, suspending agents,diluents, buffers, penetration enhancers, emulsifiers, binders,thickeners, flavoring agents (for oral administration).

Pharmaceutical Carriers for Antibodies and Peptides

More specifically with respect to high affinity MASP-3 inhibitoryantibodies, as described herein, exemplary formulations can beparenterally administered as injectable dosages of a solution orsuspension of the compound in a physiologically acceptable diluent witha pharmaceutical carrier that can be a sterile liquid such as water,oils, saline, glycerol or ethanol. Additionally, auxiliary substancessuch as wetting or emulsifying agents, surfactants, pH bufferingsubstances and the like can be present in compositions comprising MASP-3antibodies. Additional components of pharmaceutical compositions includepetroleum (such as of animal, vegetable or synthetic origin), forexample, soybean oil and mineral oil. In general, glycols such aspropylene glycol or polyethylene glycol are preferred liquid carriersfor injectable solutions.

The MASP-3 antibodies can also be administered in the form of a depotinjection or implant preparation that can be formulated in such a manneras to permit a sustained or pulsatile release of the active agents.

IV. MODES OF ADMINISTRATION

The pharmaceutical compositions comprising the MASP-3 inhibitoryantibodies, optionally in combination with MASP-2 inhibitory agents maybe administered in a number of ways depending on whether a local orsystemic mode of administration is most appropriate for the conditionbeing treated. Further, the compositions of the present invention can bedelivered by coating or incorporating the compositions on or into animplantable medical device.

Systemic Delivery

As used herein, the terms “systemic delivery” and “systemicadministration” are intended to include but are not limited to oral andparenteral routes including intramuscular (IM), subcutaneous,intravenous (IV), intraarterial, inhalational, sublingual, buccal,topical, transdermal, nasal, rectal, vaginal and other routes ofadministration that effectively result in dispersement of the deliveredagent to a single or multiple sites of intended therapeutic action.Preferred routes of systemic delivery for the present compositionsinclude intravenous, intramuscular, subcutaneous, intraarterial andinhalational. It will be appreciated that the exact systemicadministration route for selected agents utilized in particularcompositions of the present invention will be determined in part toaccount for the agent's susceptibility to metabolic transformationpathways associated with a given route of administration. For example,peptidergic agents may be most suitably administered by routes otherthan oral.

The MASP-3 inhibitory antibodies, as described herein, can be deliveredinto a subject in need thereof by any suitable means. Methods ofdelivery of MASP-3 antibodies and polypeptides include administration byoral, pulmonary, parenteral (e.g., intramuscular, intraperitoneal,intravenous (IV) or subcutaneous injection), inhalation (such as via afine powder formulation), transdermal, nasal, vaginal, rectal, orsublingual routes of administration, and can be formulated in dosageforms appropriate for each route of administration.

By way of representative example, MASP-3 inhibitory antibodies andpeptides can be introduced into a living body by application to a bodilymembrane capable of absorbing the polypeptides, for example the nasal,gastrointestinal and rectal membranes. The polypeptides are typicallyapplied to the absorptive membrane in conjunction with a permeationenhancer. (See, e.g., Lee, V. H. L., Crit. Rev. Ther. Drug Carrier Sys.5:69, (1988); Lee, V. H. L., J. Controlled Release 13:213, (1990); Lee,V. H. L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, NewYork (1991); DeBoer, A. G., et al., J. Controlled Release 13:241,(1990). For example, STDHF is a synthetic derivative of fusidic acid, asteroidal surfactant that is similar in structure to the bile salts, andhas been used as a permeation enhancer for nasal delivery. (Lee, W. A.,Biopharm. 22, Nov./Dec. 1990.)

The MASP-3 inhibitory antibodies as described herein may be introducedin association with another molecule, such as a lipid, to protect thepolypeptides from enzymatic degradation. For example, the covalentattachment of polymers, especially polyethylene glycol (PEG), has beenused to protect certain proteins from enzymatic hydrolysis in the bodyand thus prolong half-life (Fuertges, F., et al., J. Controlled Release11:139, (1990)). Many polymer systems have been reported for proteindelivery (Bae, Y. H., et al., J. Controlled Release 9:271, (1989); Hori,R., et al., Pharm. Res. 6:813, (1989); Yamakawa, I., et al., J. Pharm.Sci. 79:505, (1990); Yoshihiro, I., et al., J. Controlled Release10:195, (1989); Asano, M., et al., J. Controlled Release 9:111, (1989);Rosenblatt, J., et al., J. Controlled Release 9:195, (1989); Makino, K.,J. Controlled Release 12:235, (1990); Takakura, Y., et al., J. Pharm.Sci. 78:117, (1989); Takakura, Y., et al., J. Pharm. Sci. 78:219,(1989)).

Recently, liposomes have been developed with improved serum stabilityand circulation half-times (see, e.g., U.S. Pat. No. 5,741,516, toWebb). Furthermore, various methods of liposome and liposome-likepreparations as potential drug carriers have been reviewed (see, e.g.,U.S. Pat. No. 5,567,434, to Szoka; U.S. Pat. No. 5,552,157, to Yagi;U.S. Pat. No. 5,565,213, to Nakamori; U.S. Pat. No. 5,738,868, toShinkarenko; and U.S. Pat. No. 5,795,587, to Gao).

For transdermal applications, the MASP-3 inhibitory antibodies, asdescribed herein, may be combined with other suitable ingredients, suchas carriers and/or adjuvants. There are no limitations on the nature ofsuch other ingredients, except that they must be pharmaceuticallyacceptable for their intended administration, and cannot degrade theactivity of the active ingredients of the composition. Examples ofsuitable vehicles include ointments, creams, gels, or suspensions, withor without purified collagen. The MASP-3 inhibitory antibodies may alsobe impregnated into transdermal patches, plasters, and bandages,preferably in liquid or semi-liquid form.

The compositions of the present invention may be systemicallyadministered on a periodic basis at intervals determined to maintain adesired level of therapeutic effect. For example, compositions may beadministered, such as by subcutaneous injection, every two to four weeksor at less frequent intervals. The dosage regimen will be determined bythe physician considering various factors that may influence the actionof the combination of agents. These factors will include the extent ofprogress of the condition being treated, the patient's age, sex andweight, and other clinical factors. The dosage for each individual agentwill vary as a function of the MASP-3 inhibitory antibody or the MASP-2inhibitory agent that is included in the composition, as well as thepresence and nature of any drug delivery vehicle (e.g., a sustainedrelease delivery vehicle). In addition, the dosage quantity may beadjusted to account for variation in the frequency of administration andthe pharmacokinetic behavior of the delivered agent(s).

Local Delivery

As used herein, the term “local” encompasses application of a drug in oraround a site of intended localized action, and may include for exampletopical delivery to the skin or other affected tissues, ophthalmicdelivery, intrathecal (IT), intracerebroventricular (ICV),intra-articular, intracavity, intracranial or intravesicularadministration, placement or irrigation. Local administration may bepreferred to enable administration of a lower dose, to avoid systemicside effects, and for more accurate control of the timing of deliveryand concentration of the active agents at the site of local delivery.Local administration provides a known concentration at the target site,regardless of interpatient variability in metabolism, blood flow, etc.Improved dosage control is also provided by the direct mode of delivery.

Local delivery of a MASP-3 inhibitory antibody or a MASP-2 inhibitoryagent may be achieved in the context of surgical methods for treating adisease or condition, such as for example during procedures such asarterial bypass surgery, atherectomy, laser procedures, ultrasonicprocedures, balloon angioplasty and stent placement. For example, aMASP-3 inhibitory antibody or a MASP-2 inhibitory agent can beadministered to a subject in conjunction with a balloon angioplastyprocedure. A balloon angioplasty procedure involves inserting a catheterhaving a deflated balloon into an artery. The deflated balloon ispositioned in proximity to the atherosclerotic plaque and is inflatedsuch that the plaque is compressed against the vascular wall. As aresult, the balloon surface is in contact with the layer of vascularendothelial cells on the surface of the blood vessel. The MASP-3inhibitory antibody or MASP-2 inhibitory agent may be attached to theballoon angioplasty catheter in a manner that permits release of theagent at the site of the atherosclerotic plaque. The agent may beattached to the balloon catheter in accordance with standard proceduresknown in the art. For example, the agent may be stored in a compartmentof the balloon catheter until the balloon is inflated, at which point itis released into the local environment. Alternatively, the agent may beimpregnated on the balloon surface, such that it contacts the cells ofthe arterial wall as the balloon is inflated. The agent may also bedelivered in a perforated balloon catheter such as those disclosed inFlugelman, M. Y., et al., Circulation 85:1110-1117, (1992). See alsopublished PCT Application WO 95/23161 for an exemplary procedure forattaching a therapeutic protein to a balloon angioplasty catheter.Likewise, the MASP-3 inhibitory agent or MASP-2 inhibitory agent may beincluded in a gel or polymeric coating applied to a stent, or may beincorporated into the material of the stent, such that the stent elutesthe MASP-3 inhibitory agent or MASP-2 inhibitory agent after vascularplacement.

MASP-3 inhibitory antibodies used in the treatment of arthritides andother musculoskeletal disorders may be locally delivered byintra-articular injection. Such compositions may suitably include asustained release delivery vehicle. As a further example of instances inwhich local delivery may be desired, MASP-3 inhibitory compositions usedin the treatment of urogenital conditions may be suitably instilledintravesically or within another urogenital structure.

V. TREATMENT REGIMENS

In prophylactic applications, the pharmaceutical compositions areadministered to a subject susceptible to, or otherwise at risk of, analternative pathway associated disease or disorder, for example, analternative pathway disease or disorder selected from the groupconsisting of paroxysmal nocturnal hemoglobinuria (PNH), age-relatedmacular degeneration (AMD), ischemia-reperfusion injury, arthritis,disseminated intravascular coagulation, thrombotic microangiopathy(including hemolytic uremic syndrome (HUS), atypical hemolytic uremicsyndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP)), asthma,dense deposit disease, pauci-immune necrotizing crescenticglomerulonephritis, traumatic brain injury, aspiration pneumonia,endophthalmitis, neuromyelitis optica, Behcet's disease, multiplesclerosis, Guillain Barre Syndrome, Alzheimer's disease, Amylotrophiclateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus(SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonarydisease (COPD), C3 glomerulopathy, transplant rejection,Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemicinflammatory response syndrome (SIRS), Acute Respiratory DistressSyndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome,Atherosclerosis, IgA Nephropathy and Myasthenia Gravis., in an amountsufficient to eliminate or reduce the risk of developing symptoms of thecondition. In therapeutic applications, the pharmaceutical compositionsare administered to a subject suspected of, or already suffering from,an alternative pathway-related disease or disorder, such as analternative pathway disease or disorder selected from the groupconsisting of paroxysmal nocturnal hemoglobinuria (PNH), age-relatedmacular degeneration (AMD), ischemia-reperfusion injury, arthritis,disseminated intravascular coagulation, thrombotic microangiopathy(including hemolytic uremic syndrome (HUS), atypical hemolytic uremicsyndrome (aHUS) or thrombotic thrombocytopenic purpura (TTP)), asthma,dense deposit disease, pauci-immune necrotizing crescenticglomerulonephritis, traumatic brain injury, aspiration pneumonia,endophthalmitis, neuromyelitis optica, Behcet's disease, multiplesclerosis, Guillain Barre Syndrome, Alzheimer's disease, Amylotrophiclateral sclerosis (ALS), lupus nephritis, systemic lupus erythematosus(SLE), Diabetic retinopathy, Uveitis, Chronic obstructive pulmonarydisease (COPD), C3 glomerulopathy, transplant rejection,Graft-versus-host disease (GVHD), hemodialysis, sepsis, Systemicinflammatory response syndrome (SIRS), Acute Respiratory DistressSyndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome,Atherosclerosis, IgA Nephropathy and Myasthenia Gravis, in atherapeutically effective amount sufficient to relieve, or at leastpartially reduce, the symptoms of the condition.

In one embodiment, the pharmaceutical composition comprising a highaffinity MASP-3 inhibitory antibody is administered to a subjectsuffering from, or at risk for developing PNH. In accordance with thisthe subject's red blood cells are opsonized by fragments of C3 in theabsence of the composition, and administration of the composition to thesubject increases the survival of red blood cells in the subject. In oneembodiment, the subject exhibits one or more symptoms in the absence ofthe composition selected from the group consisting of (i) below normallevels of hemoglobin, (ii) below normal levels of platelets; (iii) abovenormal levels of reticulocytes, and (iv) above normal levels ofbilirubin, and administration of the composition to the subject improvesat least one or more of the symptoms, resulting in (i) increased,normal, or nearly normal levels of hemoglobin (ii) increased, normal ornearly normal levels of platelets, (iii) decreased, normal or nearlynormal levels of reticulocytes, and/or (iv) decreased, normal or nearlynormal levels of bilirubin.

In both prophylactic and therapeutic regimens for the treatment,prevention or reduction in severity of a disease or condition selectedfrom the group consisting of paroxysmal nocturnal hemoglobinuria (PNH),age-related macular degeneration (AMD), ischemia-reperfusion injury,arthritis, disseminated intravascular coagulation, thromboticmicroangiopathy (including hemolytic uremic syndrome (HUS), atypicalhemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura(TTP)), asthma, dense deposit disease, pauci-immune necrotizingcrescentic glomerulonephritis, traumatic brain injury, aspirationpneumonia, endophthalmitis, neuromyelitis optica and Behcet's disease,compositions comprising high affinity MASP-3 inhibitory antibodies andoptionally MASP-2 inhibitory agents may be administered in severaldosages until a sufficient therapeutic outcome has been achieved in thesubject. In one embodiment of the invention, the high affinity MASP-3inhibitory antibody and/or MASP-2 inhibitory agent may be administeredto an adult patient (e.g., an average adult weight of 70 kg) in a dosageof from 0.1 mg to 10,000 mg, more suitably from 1.0 mg to 5,000 mg, moresuitably 10.0 mg to 2,000 mg, more suitably 10.0 mg to 1,000 mg andstill more suitably from 50.0 mg to 500 mg, or 10 to 200 mg. Forpediatric patients, dosage can be adjusted in proportion to thepatient's weight.

Application of the high affinity MASP-3 inhibitory antibodies andoptional MASP-2 inhibitory compositions of the present invention may becarried out by a single administration of the composition (e.g., asingle composition comprising MASP-3 and optionally MASP-2 inhibitoryagents, or bispecific or dual inhibitory agents, or co-administration ofseparate compositions), or a limited sequence of administrations, fortreatment of an alternative pathway-related disease or disorder, such asa disease or disorder selected form the group consisting of paroxysmalnocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD),ischemia-reperfusion injury, arthritis, disseminated intravascularcoagulation, thrombotic microangiopathy (including hemolytic uremicsyndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thromboticthrombocytopenic purpura (TTP)), asthma, dense deposit disease,pauci-immune necrotizing crescentic glomerulonephritis, traumatic braininjury, aspiration pneumonia, endophthalmitis, neuromyelitis optica,Behcet's disease, multiple sclerosis, Guillain Barre Syndrome,Alzheimer's disease, Amylotrophic lateral sclerosis (ALS), lupusnephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy,Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy andMyasthenia Gravis.

Alternatively, the composition may be administered at periodic intervalssuch as daily, biweekly, weekly, every other week, monthly or bimonthlyover an extended period of time for as determined by a physician foroptimal therapeutic effect.

In some embodiments, a first composition comprising at least one highaffinity MASP-3 inhibitory antibody and a second composition comprisingat least one MASP-2 inhibitory agent are administered to a subjectsuffering from, or at risk for developing a disease or conditionselected from the group consisting of paroxysmal nocturnalhemoglobinuria (PNH), age-related macular degeneration (AMD),ischemia-reperfusion injury, arthritis, disseminated intravascularcoagulation, thrombotic microangiopathy (including hemolytic uremicsyndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thromboticthrombocytopenic purpura (TTP)), asthma, dense deposit disease,pauci-immune necrotizing crescentic glomerulonephritis, traumatic braininjury, aspiration pneumonia, endophthalmitis, neuromyelitis optica,Behcet's disease, multiple sclerosis, Guillain Barre Syndrome,Alzheimer's disease, Amylotrophic lateral sclerosis (ALS), lupusnephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy,Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy andMyasthenia Gravis.

In one embodiment, the first composition comprising at least one highaffinity MASP-3 inhibitory antibody and a second composition comprisingat least one MASP-2 inhibitory agent are administered simultaneously(i.e., within a time separation of no more than about 15 minutes orless, such as no more than any of 10, 5 or 1 minute). In one embodiment,the first composition comprising at least one high affinity MASP-3inhibitory antibody and a second composition comprising at least oneMASP-2 inhibitory agent are administered sequentially (i.e., the firstcomposition is administered either prior to or after the administrationof the second composition, wherein the time separation of administrationis more than 15 minutes). In some embodiments, the first compositioncomprising at least one high affinity MASP-3 inhibitory antibody and asecond composition comprising at least one MASP-2 inhibitory agent areadministered concurrently (i.e., the administration period of the firstcomposition overlaps with the administration of the second composition).For example, in some embodiments, the first composition and/or thesecond composition are administered for a period of at least one, two,three or four weeks or longer. In one embodiment, at least one highaffinity MASP-3 inhibitory antibody and at least one MASP-2 inhibitoryagent are combined in a unit dosage form. In one embodiment, a firstcomposition comprising at least one high affinity MASP-3 inhibitoryantibody and a second composition comprising at least one MASP-2inhibitory agent are packaged together in a kit for use in treatment ofan alternative pathway-related disease or condition, such as paroxysmalnocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD),ischemia-reperfusion injury, arthritis, disseminated intravascularcoagulation, thrombotic microangiopathy (including hemolytic uremicsyndrome (HUS), atypical hemolytic uremic syndrome (aHUS) or thromboticthrombocytopenic purpura (TTP)), asthma, dense deposit disease,pauci-immune necrotizing crescentic glomerulonephritis, traumatic braininjury, aspiration pneumonia, endophthalmitis, neuromyelitis optica,Behcet's disease, multiple sclerosis, Guillain Barre Syndrome,Alzheimer's disease, Amylotrophic lateral sclerosis (ALS), lupusnephritis, systemic lupus erythematosus (SLE), Diabetic retinopathy,Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy orMyasthenia Gravis.

In some embodiments, the subject suffering from PNH, age-related maculardegeneration (AMD), ischemia-reperfusion injury, arthritis, disseminatedintravascular coagulation, thrombotic microangiopathy (includinghemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome(aHUS) or thrombotic thrombocytopenic purpura (TTP)), asthma, densedeposit disease, pauci-immune necrotizing crescentic glomerulonephritis,traumatic brain injury, aspiration pneumonia, endophthalmitis,neuromyelitis optica, Behcet's disease, multiple sclerosis, GuillainBarre Syndrome, Alzheimer's disease, Amylotrophic lateral sclerosis(ALS), lupus nephritis, systemic lupus erythematosus (SLE), Diabeticretinopathy, Uveitis, Chronic obstructive pulmonary disease (COPD), C3glomerulopathy, transplant rejection, Graft-versus-host disease (GVHD),hemodialysis, sepsis, Systemic inflammatory response syndrome (SIRS),Acute Respiratory Distress Syndrome (ARDS), ANCA vasculitis,Anti-phospholipid syndrome, Atherosclerosis, IgA Nephropathy andMyasthenia Gravis has previously undergone, or is currently undergoingtreatment with a terminal complement inhibitor that inhibits cleavage ofcomplement protein C5. In some embodiments, the method comprisesadministering to the subject a composition of the invention comprising ahigh affinity MASP-3 inhibitory antibody and optionally a MASP-2inhibitor and further administering to the subject a terminal complementinhibitor that inhibits cleavage of complement protein C5. In someembodiments, the terminal complement inhibitor is a humanized anti-05antibody or antigen-binding fragment thereof. In some embodiments, theterminal complement inhibitor is eculizumab.

VI. EXAMPLES

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention. All literature citations herein are expressly incorporated byreference.

Example 1

This Example demonstrates that MASP-2 deficient mice are protected fromNeisseria meningitidis induced mortality after infection with either N.meningitidis serogroup A or N. meningitidis serogroup B.

Methods:

MASP-2 knockout mice (MASP-2 KO mice) were generated as described inExample 1 of U.S. Pat. No. 7,919,094, hereby incorporated herein byreference. 10-week-old MASP-2 KO mice (n=10) and wild-type (WT) C57/BL6mice (n=10) were inoculated by intraperitoneal (i.p.) injection with adosage of 2.6×10⁷ CFU of N. meningitidis serogroup A Z2491 in a volumeof 100 μl. The infective dose was administered to mice in conjunctionwith iron dextran at a final concentration of 400 mg/kg. Survival of themice after infection was monitored over a 72-hour time period.

In a separate experiment, 10-week-old MASP-2 KO mice (n=10) and WTC57/BL6 mice (n=10) were inoculated by i.p. injection with a dosage of6×10⁶ CFU of N. meningitidis serogroup B strain MC58 in a volume of 100μL. The infective dose was administered to mice in conjunction with irondextran at a final dose of 400 mg/kg. Survival of the mice afterinfection was monitored over a 72-hour time period. An illness score wasalso determined for the WT and MASP-2 KO mice during the 72-hour timeperiod after infection, based on the illness scoring parametersdescribed below in TABLE 5, which is based on the scheme of Fransen etal. (2010) with slight modifications.

TABLE 5 Illness Scoring associated with clinical signs in infected miceSigns Score Normal 0 Slightly ruffled fur 1 Ruffled fur, slow and stickyeyes 2 Ruffled fur, lethargic and eyes shut 3 Very sick and no movementafter stimulation 4 Dead 5

Blood samples were taken from the mice at hourly intervals afterinfection and analyzed to determine the serum level (log cfu/mL) of N.meningitidis in order to verify infection and determine the rate ofclearance of the bacteria from the serum.

Results:

FIG. 6 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of MASP-2 KO and WT mice after administration of an infectivedose of 2.6×10⁷ cfu of N. meningitidis serogroup A Z2491. As shown inFIG. 6, 100% of the MASP-2 KO mice survived throughout the 72-hourperiod after infection. In contrast, only 80% of the WT mice (p=0.012)were still alive 24 hours after infection, and only 50% of the WT micewere still alive at 72 hours after infection. These results demonstratethat MASP-2-deficient mice are protected from N. meningitidis serogroupA Z2491-induced mortality.

FIG. 7 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of MASP-2 KO and WT mice after administration of an infectivedose of 6×10⁶ cfu of N. meningitidis serogroup B strain MC58. As shownin FIG. 7, 90% of the MASP-2 KO mice survived throughout the 72-hourperiod after infection. In contrast, only 20% of the WT mice (p=0.0022)were still alive 24 hours after infection. These results demonstratethat MASP-2-deficient mice are protected from N. meningitidis serogroupB strain MC58-induced mortality.

FIG. 8 graphically illustrates the log cfu/mL of N. meningitidisserogroup B strain MC58 recovered at different time points in bloodsamples taken from the MASP-2 KO and WT mice after i.p. infection with6×10⁶ cfu of N. meningitidis serogroup B strain MC58 (n=3 at differenttime points for both groups of mice). The results are expressed asMeans±SEM. As shown in FIG. 8, in WT mice the level of N. meningitidisin the blood reached a peak of about 6.0 log cfu/mL at 24 hours afterinfection and dropped to about 4.0 log cfu/mL by 36 hours afterinfection. In contrast, in the MASP-2 KO mice, the level of N.meningitidis reached a peak of about 4.0 log cfu/mL at 12 hours afterinfection and dropped to about 1.0 log cfu/mL by 36 hours afterinfection (the symbol “*” indicates p<0.05; the symbol “**” indicatesp=0.0043). These results demonstrate that although the MASP-2 KO micewere infected with the same dose of N. meningitidis serogroup B strainMC58 as the WT mice, the MASP-2 KO mice have enhanced clearance ofbacteraemia as compared to WT.

FIG. 9 graphically illustrates the average illness score of MASP-2 KOand WT mice at 3, 6, 12 and 24 hours after infection with 6×10⁶ cfu ofN. meningitidis serogroup B strain MC58. As shown in FIG. 9, theMASP-2-deficient mice showed high resistance to the infection, with muchlower illness scores at 6 hours (symbol “*” indicates p=0.0411), 12hours (symbol “**” indicates p=0.0049) and 24 hours (symbol “***”indicates p=0.0049) after infection, as compared to WT mice. The resultsin FIG. 9 are expressed as means±SEM.

In summary, the results in this Example demonstrate thatMASP-2-deficient mice are protected from N. meningitides-inducedmortality after infection with either N. meningitidis serogroup A or N.meningitidis serogroup B.

Example 2

This Example demonstrates that the administration of MASP-2 antibodyafter infection with N. meningitidis increases the survival of miceinfected with N. meningitidis.

Background/Rationale:

As described in Example 24 of U.S. Pat. No. 7,919,094, incorporatedherein by reference, rat MASP-2 protein was utilized to pan a Fab phagedisplay library, from which Fab2 #11 was identified as a functionallyactive antibody. Full-length antibodies of the rat IgG2c and mouse IgG2aisotypes were generated from Fab2 #11. The full-length MASP-2 antibodyof the mouse IgG2a isotype was characterized for pharmacodynamicparameters (as described in Example 38 of U.S. Pat. No. 7,919,094).

In this Example, the mouse MASP-2 full-length antibody derived from Fab2#11 was analyzed in the mouse model of N. meningitidis infection.

Methods:

The mouse IgG2a full-length MASP-2 antibody isotype derived from Fab2#11, generated as described above, was tested in the mouse model of N.meningitidis infection as follows.

1. Administration of Mouse-MASP-2 Monoclonal Antibodies (MoAb) AfterInfection

9-week-old C57/BL6 Charles River mice were treated with inhibitory mouseMASP-2 antibody (1.0 mg/kg) (n=12) or control isotype antibody (n=10) at3 hours after i.p. injection with a high dose (4×10⁶ cfu) of N.meningitidis serogroup B strain MC58.

Results:

FIG. 10 is a Kaplan-Meyer plot graphically illustrating the percentsurvival of mice after administration of an infective dose of 4×10⁶ cfuof N. meningitidis serogroup B strain MC58, followed by administration 3hours post-infection of either inhibitory MASP-2 antibody (1.0 mg/kg) orcontrol isotype antibody. As shown in FIG. 10, 90% of the mice treatedwith MASP-2 antibody survived throughout the 72-hour period afterinfection. In contrast, only 50% of the mice treated with isotypecontrol antibody survived throughout the 72-hour period after infection.The symbol “*” indicates p=0.0301, as determined by comparison of thetwo survival curves.

These results demonstrate that administration of a MASP-2 antibody iseffective to treat and improve survival in subjects infected with N.meningitidis.

As demonstrated herein, the use of MASP-2 antibody in the treatment of asubject infected with N. meningitidis is effective when administeredwithin 3 hours post-infection, and is expected to be effective within 24hours to 48 hours after infection. Meningococcal disease (eithermeningococcemia or meningitis) is a medical emergency, and therapy willtypically be initiated immediately if meningococcal disease is suspected(i.e., before N. meningitidis is positively identified as theetiological agent).

In view of the results in the MASP-2 KO mouse demonstrated in EXAMPLE 1,it is believed that administration of MASP-2 antibody prior to infectionwith N. meningitidis would also be effective to prevent or amelioratethe severity of infection.

Example 3

This Example demonstrates the complement-dependent killing of N.meningitidis in human sera is MASP-3-dependent.

Rationale:

Patients with decreased serum levels of functional MBL display increasedsusceptibility to recurrent bacterial and fungal infections (Kilpatricket al., Biochim Biophys Acta 1572:401-413 (2002)). It is known that N.meningitidis is recognized by MBL, and it has been shown thatMBL-deficient sera do not lyse N. meningitidis.

In view of the results described in Examples 1 and 2, a series ofexperiments were carried out to determine the efficacy of administrationof MASP-2 antibody to treat N. meningitidis infection incomplement-deficient and control human sera. Experiments were carriedout in a high concentration of serum (20%) in order to preserve thecomplement pathway.

Methods:

1. Serum Bactericidal Activity in Various Complement-Deficient HumanSera and in Human Sera Treated with Human MASP-2 Antibody

The following complement-deficient human sera and control human serawere used in this experiment:

TABLE 6 Human serum samples tested (as shown in FIG. 11) Sample Serumtype A Normal human sera (NHS) + human MASP-2 Ab B NHS + isotype controlAb C MBL −/− human serum D NHS E Heat-Inactivated (HI) NHS

A recombinant antibody against human MASP-2 was isolated from acombinatorial Antibody Library (Knappik, A., et al., J. Mol. Biol.296:57-86 (2000)), using recombinant human MASP-2A as an antigen (Chen,C. B. and Wallis, J. Biol. Chem. 276:25894-25902 (2001)). An anti-humanscFv fragment that potently inhibited lectin pathway-mediated activationof C4 and C3 in human plasma (IC50-20 nM) was identified and convertedto a full-length human IgG4 antibody.

N. meningitidis serogroup B-MC58 was incubated with the different serashow in TABLE 6, each at a serum concentration of 20%, with or withoutthe addition of inhibitory human MASP-2 antibody (3 μg in 100 μl totalvolume) at 37° C. with shaking. Samples were taken at the following timepoints: 0-, 30-, 60- and 90-minute intervals, plated out and then viablecounts were determined. Heat-inactivated human serum was used as anegative control.

Results:

FIG. 11 graphically illustrates the log cfu/mL of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in thehuman sera samples shown in TABLE 6. TABLE 7 provides the Student'st-test results for FIG. 11.

TABLE 7 Student's t-test Results for FIG. 11 (time point 60 minutes)Significant? Mean Diff. (Log) P < 0.05? P value summary A vs B −0.3678Yes ***(0.0002)    A vs C −1.1053 Yes ***(p < 0.0001) A vs D −0.2111 Yes**(0.0012)   C vs D 1.9 Yes ***(p < 0.0001)

As shown in FIG. 11 and TABLE 7, complement-dependent killing of N.meningitidis in human 20% serum was significantly enhanced by theaddition of the human MASP-2 inhibitory antibody.

2. Serum Bactericidal Activity in Various Complement-Deficient HumanSera

The following complement-deficient human sera and control human serawere used in this experiment:

TABLE 8 Human serum samples tested (as shown in FIG. 12) Sample SerumType A Normal human serum (NHS) B Heat-inactivated NHS C MBL−/− DMASP-3−/− (MASP-1+) Note: The MASP-3−/− (MASP-1+) serum in sample D wastaken from a subject with 3MC syndrome, which is a unifying term for theoverlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. Asfurther described in Example 4, the mutations in exon 12 of the MASP-1/3gene render the serine protease domain of MASP-3, but not MASP-1dysfunctional. As described in Example 10, pro-factor D ispreferentially present in 3MC serum, whereas activated factor D ispreferentially present in normal human serum.

N. meningitidis serogroup B-MC58 was incubated with differentcomplement-deficient human sera, each at a serum concentration of 20%,at 37° C. with shaking. Samples were taken at the following time points:0-, 15-, 30-, 45-, 60-, 90- and 120-minute intervals, plated out andthen viable counts were determined. Heat-inactivated human serum wasused as a negative control.

Results:

FIG. 12 graphically illustrates the log cfu/mL of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in thehuman sera samples shown in TABLE 8. As shown in FIG. 12, the WT (NHS)serum has the highest level of bactericidal activity for N.meningitidis. In contrast, the MBL −/− and MASP-3 −/− (which isMASP-1-sufficient) human sera do not have any bactericidal activity.These results indicate that complement-dependent killing of N.meningitidis in human 20% (v/v) serum is MASP-3- and MBL-dependent.TABLE 9 provides the Student's t-test results for FIG. 12.

TABLE 9 Student's t-test Results for FIG. 12 Time Mean Point Diff.Significant? P value Comparison (min) (Log) P < 0.05? Summary A vs B 60−0.8325 Yes ***(p < 0.0001) A vs B 90 −1.600 Yes ***(p < 0.0001) A vs C60 −1.1489 Yes ***(p < 0.0001) A vs C 90 −1.822 Yes ***(p < 0.0001) A vsD 60 −1.323 Yes ***(0.0005)    A vs D 90 −2.185 Yes ***(p < 0.0001)

In summary, the results shown in FIG. 12 and TABLE 9 demonstrate thatcomplement-dependent killing of N. meningitidis in 20% human serum isMASP-3- and MBL-dependent.

3. Complement-Dependent Killing of N. meningitidis in 20% (v/v) MouseSera Deficient of MASP-2, MASP-1/3 or MBL A/C.

The following complement-deficient mouse sera and control mouse serawere used in this experiment:

TABLE 10 Mouse serum samples tested (as shown in FIG. 13) Sample SerumType A WT B MASP-2−/− C MASP-1/3−/− D MBL A/C−/− E WT heat-inactivated(HIS)

N. meningitidis serogroup B-MC58 was incubated with differentcomplement-deficient mouse sera, each at a serum concentration of 20%,at 37° C. with shaking. Samples were taken at the following time points:0-, 15-, 30-, 60-, 90- and 120-minute intervals, plated out and thenviable counts were determined. Heat-inactivated human serum was used asa negative control.

Results:

FIG. 13 graphically illustrates the log cfu/mL of viable counts of N.meningitidis serogroup B-MC58 recovered at different time points in themouse serum samples shown in TABLE 10. As shown in FIG. 13, the MASP-2−/−mouse sera have a higher level of bactericidal activity for N.meningitidis than WT mouse sera. In contrast, the MASP-1/3 −/−mouse serado not have any bactericidal activity. The symbol “**” indicatesp=0.0058, the symbol “***” indicates p=0.001. TABLE 11 provides theStudent's t-test results for FIG. 13.

TABLE 11 Student's t-test Results for FIG. 13 Mean Diff. Significant? Pvalue Comparison Time point (LOG) (p < 0.05)? summary A vs. B 60 min.0.39 yes ** (0.0058) A vs. B 90 min. 0.6741 yes *** (0.001) 

In summary, the results in this Example demonstrate that MASP-2 −/−serumhas a higher level of bactericidal activity for N. meningitidis than WTserum and that complement-dependent killing of N. meningitidis in 20%serum is MASP-3- and MBL-dependent.

Example 4

This Example describes a series of experiments that were carried out todetermine the mechanism of the MASP-3-dependent resistance to N.meningitidis infection observed in MASP-2 KO mice, as described inExamples 1-3.

Rationale:

In order to determine the mechanism of MASP-3-dependent resistance to N.meningitidis infection observed in MASP-2 KO mice (described in Examples1-3 above), a series of experiments were carried out as follows.

1. MASP-1/3-Deficient Mice are not Deficient of Lectin PathwayFunctional Activity (also Referred to as “LEA-2”)

Methods:

In order to determine whether MASP-1/3-deficient mice are deficient oflectin pathway functional activity (also referred to as LEA-2), an assaywas carried out to measure the kinetics of C3 convertase activity inplasma from various complement-deficient mouse strains tested underlectin activation pathway-specific assay conditions (1% plasma), asdescribed in Schwaeble W. et al., PNAS vol 108(18):7523-7528 (2011),hereby incorporated herein by reference.

Plasma was tested from WT, C4−/−, MASP-1/3−/−; Factor B−/−, andMASP-2−/− mice as follows.

To measure C3 activation, microtiter plates were coated with mannan (1μg/well), zymosan (1 μg/well) in coating buffer (15 mM Na₂Co₃, 35 mMNaHCO₃), or immune complexes, generated in situ by coating with 1% humanserum albumin (HSA) in coating buffer then adding sheep anti-HAS serum(2 μg/mL) in TBS (10mM Tris, 140 mM NaCl, pH 7.4) with 0.05% Tween 20and 5 mM Ca⁺⁺. Plates were blocked with 0.1% HSA in TBS and washed threetimes with TBS/Tween20/Ca⁺⁺. Plasma samples were diluted in 4 mMbarbital, 145 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4, added to theplates and incubated for 1.5 h at 37° C. After washing, bound C3b wasdetected using rabbit anti-human C3c (Dako), followed by alkalinephosphatase-conjugated goat anti-rabbit IgG and p-nitrophenyl phosphate.

Results:

The kinetics of C3 activation (as measured by C3b deposition onmannan-coated plates with 1% serum) under lectin pathway-specificconditions is shown in FIG. 14. No C3 cleavage was seen in MASP-2−/−plasma. Factor B−/− (Factor B −/−) plasma cleaved C3 at half the rate ofWT plasma, likely due to the loss of the amplification loop. Asignificant delay in the lectin pathway-dependent conversion of C3 toC3b was seen in C4−/− (T_(1/2)=33 min) as well as inMASP-1/3−/−deficient plasma (T1/2=49 min). This delay of C3 activationin MASP-1/3−/−plasma has been shown to be MASP-1- rather thanMASP-3-dependent. (See Takahashi M. et al., J. Immunol 180:6132-6138(2008)). These results demonstrate that MASP-1/3-deficient mice are notdeficient of lectin pathway functional activity (also referred to as“LEA-2”).

2. Effect of Hereditary MASP-3 Deficiency on Alternative PathwayActivation.

Rationale:

The effect of hereditary MASP-3 deficiency on alternative pathwayactivation was determined by testing serum of a MASP-3-deficient patientwith 3MC syndrome caused by a frame-shift mutation in the exon encodingthe serine protease of MASP-3. The 3MC syndrome is a unifying term forthe overlapping Carneavale, Mingarelli, Malpuech and Michels syndromes.These rare autosomal recessive disorders exhibit a spectrum ofdevelopmental features, including characteristic facial dysmorphism,cleft lip and/or palate, craniosynostosis, learning disability andgenital, limb and vesicorenal abnormalities. Rooryck et al., NatureGenetics 43:197-203 (2011) studied 11 families with 3MC syndrome andidentified two mutated genes, COLEC11 and MASP-1. The mutations in theMASP-1 gene render the exon encoding the serine protease domain ofMASP-3, but not the exons encoding the serine protease of MASP-1,dysfunctional. Therefore, 3MC patients with mutations in the exonencoding the serine protease of MASP-3 are deficient of MASP-3 butsufficient in MASP-1.

Methods:

MASP-3-deficient serum was obtained from a 3MC patient, the mother andfather of the 3MC patient (both heterozygous for the allele bearing amutation that renders the exon encoding the MASP-3 serine proteasedomain dysfunctional), as well as from a C4-deficient patient (deficientin both human C4 genes) and an MBL-deficient subject. An alternativepathway assay was carried out under traditional AP-specific conditions(BBS/ Mg⁺⁺/EGTA, without Ca⁺⁺, wherein BBS=barbital buffered salinecontaining sucrose), as described in Bitter-Suermann et al., Eur. J.Immunol 11:291-295 (1981)), on zymosan-coated microtiter plates at serumconcentrations ranging from 0.5 to 25% and C3b deposition was measuredover time.

Results:

FIG. 15 graphically illustrates the level of alternative pathway-drivenC3b deposition on zymosan-coated microtiter plates as a function ofserum concentration in serum samples obtained from MASP-3-deficient,C4-deficient and MBL-deficient subjects. As shown in FIG. 15,MASP-3-deficient patient serum has residual alternative pathway (AP)activity at high serum concentrations (25%, 12.5%, 6.25% serumconcentrations), but a significantly higher AP₅₀ (i.e., 9.8% of serumneeded to achieve 50% of maximum C3 deposition).

FIG. 16 graphically illustrates the level of alternative pathway-drivenC3b deposition on zymosan-coated microtiter plates under “traditional”alternative pathway-specific (AP-specific) conditions (i.e.,BBS/EGTA/Mg⁺⁺ without Ca⁺⁺) as a function of time in 10% human serumsamples obtained from MASP-3-deficient, C4-deficient and MBL-deficienthuman subjects.

TABLE 12 below summarizes the AP₅₀ results shown in FIG. 15 and thehalf-times for C3b deposition shown in FIG. 16.

TABLE 12 Summary of Results shown in FIGS. 15 and 16 Serum type AP₅₀ (%)T_(1/2) (min) MASP-3-deficient 9.8 37.4 (3MC patient) Mother of 3MCpatient 4.3 17.2 (heterozygous) Father of 3MC patient 4.3 20.9(heterozygous) C4-deficient 4.0 11.6 MBL-deficient 4.8 11.0 Note: InBBS/Mg⁺⁺/EGTA buffer, the lectin pathway-mediated effects are deficientdue to absence of Ca⁺⁺ in this buffer.

In summary, under the conditions of these assays, the alternativepathway is significantly compromised in the 3MC patient.

3. Measurement of C3b Deposition on Mannan, Zymosan and S. pneumonia D39in Mouse Sera Deficient of MASP-2 or MASP-1/3.

Methods:

C3b deposition was measured on mannan, zymosan and S. pneumoniaD39-coated microtiter plates using mouse serum concentrations rangingfrom 0% to 20% obtained from MASP-2−/−, MASP-1/3−/−and WT mice. The C3bdeposition assays were carried out under either “traditional”alternative pathway-specific conditions (i.e. BBS/EGTA/Mg⁺⁺ withoutCa⁺⁺), or under physiological conditions allowing both the lectinpathway and the alternative pathway to function (i.e., BBS/Mg⁺⁺/Ca⁺⁺).

Results:

FIG. 17A graphically illustrates the level of C3b deposition onmannan-coated microtiter plates as a function of serum concentration inserum samples obtained from WT, MASP-2-deficient, and MASP-1/3-deficientmice under traditional alternative pathway-specific conditions (i.e.,BBS/EGTA/Mg⁺⁺ without Ca⁺⁺), or under physiological conditions allowingboth the lectin pathway and the alternative pathway to function(BBS/Mg⁺⁺/Ca⁺⁺). FIG. 17B graphically illustrates the level of C3bdeposition on zymosan-coated microtiter plates as a function of serumconcentration in serum samples from WT, MASP-2-deficient, andMASP-1/3-deficient mice under traditional AP-specific conditions (i.e.,BBS/EGTA/Mg⁺⁺ without Ca⁺⁺), or under physiological conditions allowingboth the lectin pathway and the alternative pathway to function(BBS/Mg⁺⁺/Ca⁺⁺). FIG. 17C graphically illustrates the level of C3bdeposition on S. pneumoniae D39-coated microtiter plates as a functionof serum concentration in serum samples from WT, MASP-2-deficient, andMASP-1/3-deficient mice under traditional AP-specific conditions (i.e.,BBS/EGTA/Mg⁺⁺ without Ca⁺⁺), or under physiological conditions allowingboth the lectin pathway and the alternative pathway to function(BBS/Mg⁺⁺/Ca⁺⁺).

FIG. 18A graphically illustrates the results of a C3b deposition assayin highly diluted sera carried out on mannan-coated microtiter platesunder traditional AP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺ withoutCa⁺⁺) or under physiological conditions allowing both the lectin pathwayand the alternative pathway to function (BBS/Mg⁺⁺/Ca⁺⁺), using serumconcentrations ranging from 0% up to 1.25%. FIG. 18B graphicallyillustrates the results of a C3b deposition assay carried out onzymosan-coated microtiter plates under traditional AP-specificconditions (i.e. BBS/EGTA/Mg⁺⁻ without Ca⁺⁺) or under physiologicalconditions allowing both the lectin pathway and the alternative pathwayto function (BBS/EGTA/Mg⁺⁺/Ca⁺⁺), using serum concentrations rangingfrom 0% up to 1.25%. FIG. 18C graphically illustrates the results of aC3b deposition assay carried out on S. pneumoniae D39-coated microtiterplates under traditional AP-specific conditions (i.e. BBS/EGTA/Mg⁺⁺without Ca⁺⁺) or under physiological conditions allowing both the lectinpathway and the alternative pathway to function (BBS/EGTA/Mg⁺⁺/Ca⁺⁺),using serum concentrations ranging from 0% up to 1.25%.

As shown in FIGS. 18A-C, C3b deposition assays were also carried outunder traditional alternative pathway-specific conditions (i.e.BBS/EGTA/Mg⁺⁺ without Ca⁺⁺) or under physiological conditions allowingboth the lectin pathway and the alternative pathway to function(BBS/Mg⁺⁺/Ca⁺⁺), using higher dilutions ranging from 0% up to 1.25%serum on mannan-coated plates (FIG. 18A); zymosan-coated plates (FIG.18B) and S. pneumoniae D39-coated plates (FIG. 18C). The alternativepathway tails off under higher serum dilutions, so the activity observedin the MASP-1/3-deficient serum in the presence of Ca⁺⁺ isMASP-2-mediated LP activity, and the activity in MASP-2-deficient serumin the presence of Ca⁺⁺is MASP-1/3-mediated residual activation of theAP.

Discussion:

The results described in this Example demonstrate that a MASP-2inhibitor (or MASP-2 KO) provides significant protection from N.meningitidis infection by promoting MASP-3-driven alternative pathwayactivation. The results of the mouse serum bacteriolysis assays and thehuman serum bacteriolysis assays further show, by monitoring the serumbactericidal activity against N. meningitidis that bactericidal activityagainst N. meningitidis is absent in MBL-deficient (mouse MBL A and MBLC double-deficient and human MBL-deficient sera).

FIG. 1 illustrates the new understanding of the lectin pathway andalternative pathway based on the results provided herein. FIG. 1delineates the role of LEA-2 in both opsonization and lysis. WhileMASP-2 is the initiator of “downstream” C3b deposition (and resultantopsonization) in multiple lectin-dependent settings physiologically(FIGS. 18A, 18B, 18C), it also plays a role in lysis of serum-sensitivebacteria. As illustrated in FIG. 1, the proposed molecular mechanismresponsible for the increased bactericidal activity of MASP-2-deficientor MASP-2-depleted serum/plasma for serum-sensitive pathogens such as N.meningitidis is that, for the lysis of bacteria, lectin pathwayrecognition complexes associated with MASP-1 and MASP-3 have to bind inclose proximity to each other on the bacterial surface, thereby allowingMASP-1 to cleave MASP-3. In contrast to MASP-1 and MASP-2, MASP-3 is notan auto-activating enzyme, but, in many instances, requiresactivation/cleavage by MASP-1 to be converted into its enzymaticallyactive form.

As further shown in FIG. 1, activated MASP-3 can then cleave C3b-boundfactor B on the pathogen surface to initiate the alternative pathwayactivation cascade by formation of the enzymatically active alternativepathway C3 and C5 convertase C3bBb and C3bBb(C3b)n, respectively.MASP-2-bearing lectin-pathway activation complexes have no part in theactivation of MASP-3 and, in the absence or after depletion of MASP-2,all-lectin pathway activation complexes will either be loaded withMASP-1 or MASP-3. Therefore, in the absence of MASP-2, the likelihood ismarkedly increased that on the microbial surface MASP-1 andMASP-3-bearing lectin-pathway activation complexes will come to sit inclose proximity to each other, leading to more MASP-3 being activatedand thereby leading to a higher rate of MASP-3-mediated cleavage ofC3b-bound factor B to form the alternative pathway C3 and C5 convertasesC3bBb and C3bBb(C3b)n on the microbial surface. This leads to theactivation of the terminal activation cascades C5b-C9 that forms theMembrane Attack Complex, composed of surface-bound C5b associated withC6, C5bC6 associated with C7, C5bC6C7 associated with C8, and C5bC6C7C8,leading to the polymerization of C9 that inserts into the bacterialsurface structure and forms a pore in the bacterial wall, which willlead to osmolytic killing of the complement-targeted bacterium.

The core of this novel concept is that the data provided herein clearlyshow that the lectin-pathway activation complexes drive the two distinctactivation routes, as illustrated in FIG. 1.

Example 5

This Example demonstrates the inhibitory effect of MASP-2 deficiencyand/or MASP-3 deficiency on lysis of red blood cells from blood samplesobtained from a mouse model of paroxysmal nocturnal hemoglobinuria(PNH).

Background/Rationale:

Paroxysmal nocturnal hemoglobinuria (PNH), also referred to asMarchiafava-Micheli syndrome, is an acquired, potentiallylife-threatening disease of the blood, characterized bycomplement-induced intravascular hemolytic anemia. The hallmark of PNHis the chronic complement-mediated intravascular hemolysis that is aconsequence of unregulated activation of the alternative pathway ofcomplement due to the absence of the complement regulators CD55 and CD59on PNH erythrocytes, with subsequent hemoglobinuria and anemia.Lindorfer, M. A., et al., Blood 115(11) (2010), Risitano, A. M,Mini-Reviews in Medicinal Chemistry, 11:528-535 (2011). Anemia in PNH isdue to destruction of red blood cells in the bloodstream. Symptoms ofPNH include red urine, due to appearance of hemoglobin in the urine,back pain, fatigue, shortness of breath and thrombosis. PNH may developon its own, referred to as “primary PNH” or in the context of other bonemarrow disorders such as aplastic anemia, referred to as “secondaryPNH”. Treatment for PNH includes blood transfusion for anemia,anticoagulation for thrombosis and the use of the monoclonal antibodyeculizumab (Soliris®), which protects blood cells against immunedestruction by inhibiting the complement system (Hillmen P. et al., N.Engl. J. Med. 350(6):552-9 (2004)). Eculizumab (Soliris®) is a humanizedmonoclonal antibody that targets the complement component C5, blockingits cleavage by C5 convertases, thereby preventing the production of C5aand the assembly of MAC. Treatment of PNH patients with eculizumab hasresulted in a reduction of intravascular hemolysis, as measured bylactate dehydrogenase (LDH), leading to hemoglobin stabilization andtransfusion independence in about half of the patients (Hillmen P, etal., Mini-Reviews in Medicinal Chemistry, vol 11(6) (2011)). Whilenearly all patients undergoing therapy with eculizumab achieve normal oralmost normal LDH levels (due to control of intravascular hemolysis),only about one third of the patients reach a hemoglobin value about 11gr/dL, and the remaining patients on eculizumab continue to exhibitmoderate to severe (i.e., transfusion-dependent) anemia, in about equalproportions (Risitano A. M. et al., Blood 113:4094-100 (2009)). Asdescribed in Risitano et al., Mini-Reviews in Medicinal Chemistry11:528-535 (2011), it was demonstrated that PNH patients on eculizumabcontained C3 fragments bound to a substantial portion of their PNHerythrocytes (while untreated patients did not), leading to theconclusion that membrane-bound C3 fragments work as opsonins on PNHerythrocytes, resulting in their entrapment in the reticuloendothelialcells through specific C3 receptors and subsequent extravascularhemolysis. Therefore, therapeutic strategies in addition to the use ofeculizumab are needed for those patients developing C3 fragment-mediatedextravascular hemolysis because they continue to require red celltransfusions.

This Example describes methods to assess the effect of MASP-2- andMASP-3-deficient serum on lysis of red blood cells from blood samplesobtained from a mouse model of PNH and demonstrates the efficacy ofMASP-2 inhibition and/or MASP-3 inhibition to treat subjects sufferingfrom PNH, and also supports the use of inhibitors of MASP-2 and/orinhibitors of MASP-3 (including dual or bispecific MASP-2/MASP-3inhibitors) to ameliorate the effects of C3 fragment-mediatedextravascular hemolysis in PNH subjects undergoing therapy with a C5inhibitor such as eculizumab.

Methods:

PNH Animal Model:

Blood samples were obtained from gene-targeted mice with deficiencies ofCrry and C3 (Crry/C3−/−) and CD55/CD59-deficient mice. These mice aremissing the respective surface complement regulators on theirerythrocytes and these erythrocytes are, therefore, susceptible tospontaneous complement autolysis as are PNH human blood cells.

In order to sensitize these erythrocytes even more, these cells wereused with and without coating by mannan and then tested for hemolysis inWT C56/BL6 plasma, MBL null plasma, MASP-2 −/− plasma, MASP-1/3 −/−plasma, human NHS, human MBL −/− plasma, and NHS treated with humanMASP-2 antibody.

1. Hemolysis Assay of Crry/C3 and CD55/CD59 Double-Deficient MurineErythrocytes in MASP-2-Deficient/Depleted Sera and Controls

Day 1. Preparation of Murine RBC (±Mannan Coating).

Materials included: fresh mouse blood, BBS/Mg⁺⁺/Ca⁺⁻ (4.4 mM barbituricacid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5mM Mg⁺⁺, 5 mMCa⁺⁺), chromium chloride, CrCl₃.6H₂O (0.5 mg/mL in BBS/Mg⁺⁺/Ca⁺⁺) andmannan, 100 μg/mL in BBS/Mg⁺⁺/Ca⁺⁺.

Whole blood (2 mL) was spun down for 1-2 min at 2000×g in a refrigeratedcentrifuge at 4° C. The plasma and buffy coat were aspirated off. Thesample was then washed 3× by re-suspending RBC pellet in 2 mL ice-coldBBS/gelatin/Mg⁺⁺/Ca⁺⁺ and repeating centrifugation step. After the thirdwash, the pellet was re-suspended in 4 mL BB S/Mg⁺⁺/Ca⁺⁺. A 2 mL aliquotof the RBC was set aside as an uncoated control. To the remaining 2 mL,2 mL CrCl3 and 2 mL mannan were added and the sample was incubated withgentle mixing at RT for 5 minutes. The reaction was terminated by adding7.5 mL BBS/gelatin/Mg⁺⁺/Ca⁺⁺. The sample was spun down as above,re-suspended in 2 mL BBS/gelatin/Mg⁺⁺/Ca⁺⁺ and washed a further twotimes as above, then stored at 4° C.

Day 2. Hemolysis Assay

Materials included BBS/gelatin/Mg⁺⁺/Ca⁺⁺ (as above), test sera, 96-wellround-bottomed and flat-bottomed plates and a spectrophotometer thatreads 96-well plates at 410-414 nm.

The concentration of the RBC was first determined and the cells wereadjusted to 10⁹/mL, and stored at this concentration. Before use, thecells were diluted in assay buffer to 10⁸/mL, and then 100 μL per wellwas used. Hemolysis was measured at 410-414 nm (allowing for greatersensitivity than 541nm). Dilutions of test sera were prepared inice-cold BBS/gelatin/Mg⁺⁺/Ca⁺⁺. 100 μL of each serum dilution waspipetted into round-bottomed plate. 100 μL of appropriately diluted RBCpreparation was added (i.e., 10⁸/mL), incubated at 37° C. for about 1hour, and observed for lysis. (The plates may be photographed at thispoint.) The plate was then spun down at maximum speed for 5 minutes. 100μL of the fluid phase was aspirated, transferred to flat-bottom plates,and the OD was recorded at 410-414 nm. The RBC pellets were retained(these can be subsequently lysed with water to obtain an inverseresult).

Experiment #1

Fresh blood was obtained from CD55/CD59 double-deficient mice and bloodof Crry/C3 double-deficient mice and erythrocytes were prepared asdescribed in detail in the above protocol. The cells were split and halfof the cells were coated with mannan and the other half were leftuntreated, adjusting the final concentration to 10⁸/mL, of which 100 μLwas used in the hemolysis assay, which was carried out as describedabove.

Results of Experiment #1: The Lectin Pathway is Involved in ErythrocyteLysis in the PNH Animal Model

In an initial experiment, it was determined that non-coated WT mouseerythrocytes were not lysed in any mouse serum. It was furtherdetermined that mannan-coated Crry−/− mouse erythrocytes were slowlylysed (more than 3 hours at 37 degrees) in WT mouse serum, but they werenot lysed in MBL null serum. (Data not shown).

It was determined that mannan-coated Crry−/−mouse erythrocytes wererapidly lysed in human serum but not in heat-inactivated NHS.Importantly, mannan-coated Crry−/− mouse erythrocytes were lysed in NHSdiluted down to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320 and 1/640dilutions all lysed). (Data not shown). In this dilution, thealternative pathway does not work (AP functional activity issignificantly reduced below 8% serum concentration).

Conclusions from Experiment #1

Mannan-coated Crry−/−mouse erythrocytes are very well lysed in highlydiluted human serum with MBL but not in that without MBL. The efficientlysis in every serum concentration tested implies that the alternativepathway is not involved or needed for this lysis. The inability ofMBL-deficient mouse serum and human serum to lyse the mannan-coatedCrry−/− mouse erythrocytes indicates that the classical pathway also hasnothing to do with the lysis observed. As lectin pathway recognitionmolecules are required (i.e., MBL), this lysis is mediated by the lectinpathway.

Experiment #2

Fresh blood was obtained from the Crry/C3 and CD55/CD59 double-deficientmice and mannan-coated Crry−/− mouse erythrocytes were analyzed in thehaemolysis assay as described above in the presence of the followinghuman serum: MASP-3 −/−; MBL null; WT; NHS pretreated with human MASP-2antibody; and heat-inactivated NHS as a control.

Results of Experiment #2: MASP-2 Inhibitors and MASP-3 DeficiencyPrevents Erythrocyte Lysis in PNH Animal Model

With the mannan-coated Crry−/− mouse erythrocytes, NHS was incubated inthe dilutions diluted down to 1/640 (i.e., 1/40, 1/80, 1/160, 1/320 and1/640), human MBL−/− serum, human MASP-3-deficient serum (from 3MCpatient), and NHS pretreated with MASP-2 mAb, and heat-inactivated NHSas a control.

The ELISA microtiter plate was spun down and the non-lysed erythrocyteswere collected on the bottom of the round-well plate. The supernatant ofeach well was collected and the amount of hemoglobin released from thelysed erythrocytes was measured by reading the OD415 nm in an ELISAreader.

It was observed that MASP-3−/− serum did not lyse mannan-coated mouseerythrocytes at all. In the control heat-inactivated NHS (negativecontrol), as expected, no lysis was observed. MBL−/− human serum lysedmannan-coated mouse erythrocytes at ⅛ and 1/16 dilutions.MASP-2-antibody-pretreated NHS lysed mannan-coated mouse erythrocytes at⅛ and 1/16 dilutions while WT human serum lysed mannan-coated mouseerythrocytes down to dilutions of 1/32.

FIG. 19 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed mouse erythrocytes (Crry/C3−/−) into the supernatantmeasured by photometry) of mannan-coated murine erythrocytes by humanserum over a range of serum dilutions in serum from MASP-3−/−,heat-inactivated (HI) NHS, MBL−/−, NHS pretreated with MASP-2 antibody,and NHS control.

FIG. 20 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed mouse erythrocytes (Crry/C3−/−) into the supernatantmeasured by photometry) of mannan-coated3 murine erythrocytes by humanserum over a range of serum concentration in serum from MASP-3−/−,heat-inactivated (HI) NHS, MBL−/−, NHS pretreated with MASP-2 antibody,and NHS control.

From the results shown in FIGS. 19 and 20, it is demonstrated thatinhibiting MASP-3 will prevent any complement-mediated lysis ofsensitized erythrocytes with deficient protection from autologouscomplement activation. MASP-2 inhibition with MASP-2 antibodysignificantly shifted the CH₅₀ and was protective to some extent, butMASP-3 inhibition was more effective.

Experiment #3

Non-coated Crry−/− mouse erythrocytes obtained from fresh blood from theCrry/C3 and CD55/CD59 double-deficient mice were analyzed in thehemolysis assay as described above in the presence of the followingsera: MASP-3−/−; MBL−/−; WT; NHS pretreated with human MASP-2 antibody,and heat-inactivated NHS as a control.

Results:

FIG. 21 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed WT mouse erythrocytes into the supernatant measured byphotometry) of non-coated murine erythrocytes over a range of serumconcentrations in human sera from a 3MC (MASP-3−/−) patient, heatinactivated (HI) NHS, MBL−/−, NHS pretreated with MASP-2 antibody, andNHS control. As shown in FIG. 21 and summarized in TABLE 13, it isdemonstrated that inhibiting MASP-3 inhibits complement-mediated lysisof non-sensitized WT mouse erythrocytes.

FIG. 22 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed mouse erythrocytes (CD55/59 −/−) into the supernatantmeasured by photometry) of non-coated murine erythrocytes by human serumover a range of serum concentrations in human sera from heat-inactivated(HI) NHS, MBL−/−, NHS pretreated with MASP-2 antibody, and NHS control.As shown in FIG. 22 and summarized in TABLE 13, it is demonstrated thatinhibiting MASP-2 was protective to a limited extent.

TABLE 13 CH₅₀ values expressed as serum concentrations Serum WTCD55/59−/− 3MC patient No lysis No lysis Heat-inactivated NHS No lysisNo lysis MBL AO/XX donor 7.2% 2.1% (MBL deficient) NHS + MASP-2 antibody5.4% 1.5% NHS 3.1% 0.73% Note: “CH₅₀” is the point at whichcomplement-mediated hemolysis reachs 50%.

In summary, the results in this Example demonstrate that inhibitingMASP-3 prevents any complement lysis of sensitized and non-sensitizederythrocytes with deficient protection from autologous complementactivation. MASP-2 inhibition also is protective to some extent.Therefore, MASP-2 and MASP-3 inhibitors alone or in combination (i.e.,co-administered, administered sequentially) or MASP-2/MASP-3 bispecificor dual inhibitors may be used to treat subjects suffering from PNH, andmay also be used to ameliorate (i.e., inhibit, prevent or reduce theseverity of) extravascular hemolysis in PNH patients undergoingtreatment with a C5 inhibitor such as eculizumab (Soliris®).

Example 6

This Example describes a hemolysis assay testing mannan-coated rabbiterythrocytes for lysis in the presence of WT or MASP-1/3−/− mouse sera.

Methods:

1.Hemolysis Assay of Rabbit RBC (Mannan Coated) in MouseMASP-1/3-Deficient Sera and WT Control Sera

Day 1. Preparation of Rabbit RBC.

Materials included: fresh rabbit blood, BBS/ Mg⁺⁺/Ca⁻⁺ (4.4 mMbarbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mMMg⁺⁺, 5 mM Ca⁺⁺), BBS/ Mg⁺⁺/Ca⁺⁺ with 0.1% gelatin, chromium chloridecontained in buffer; i.e., CrCl₃.6 H₂O (0.5 mg /mL in BBS/Mg⁺⁺/Ca⁺⁺) andmannan, 100 μg/mL in BBS/ Mg⁺⁺/Ca⁺⁺.

1. Rabbit whole blood (2 mL) was split into two 1.5 mL eppendorf tubesand centrifuged for 3 minutes at 8000 rpm (approximately 5.9 rcf) in arefrigerated eppendorf centrifuge at 4° C. The RBC pellet was washedthree times after re-suspending in ice-cold BBS/Mg⁺⁺/Ca⁺⁺. After thethird wash, the pellet was re-suspended in 4 mL BBS/Mg⁺⁺/Ca⁺⁺. Two mL ofthis aliquot were added to a 15-mL falcon tube to be used as theuncoated control. The remaining 2 mL of the RBCs aliquot were diluted in2 mL of CrCl₃ buffer, 2 mL of the mannan solution were added and thesuspension was incubated at room temperature for 5 minutes with gentlemixing. The reaction was terminated by adding 7.5 mL of BBS/0.1%gelatin/Mg⁺⁺/Ca⁺⁺ to the mixture. The erythrocytes were pelleted and theRBCs were washed twice with BBS/0.1% gelatin/Mg⁺⁺/Ca⁺⁺ as describedabove. The RBCs suspension was stored in BBS/0.1% gelatin/ Mg⁺⁺/Ca⁺⁺ at4° C.

2. 100 μL of suspended RBCs were diluted with 1.4 mL water and spun downat 8000 rpm (approximately 5.9 rcf) for 3 minutes and the OD of thesupernatant was adjusted to 0.7 at 541 nm (an OD of 0.7 at 541 nmcorresponds to approximately 10⁹ erythrocytes/mL).

3. The re-suspended RBCs were diluted with BBS/0.1% gelatin/Mg⁻⁺/Ca⁺⁺ toa concentration of 10⁸ /mL.

4. Dilutions of the test sera were prepared in ice-cold BBS/gelatin/Mg⁺⁺/Ca⁺⁺ and 100 μL of each serum dilution were pipetted into thecorresponding well of round-bottom plate. 100 μL of appropriatelydiluted RBC (108/mL) were added to each well. As a control for completelysis, purified water (100 μL) was mixed with the diluted RBC (100 μL)to cause 100% lysis, while BBS/0.1% gelatin/ Mg⁺⁺/Ca⁻⁺ without serum(100 μL) was used as a negative control. The plate was then incubatedfor 1 hour at 37° C.

5. The round-bottom plate was centrifuged at 3250 rpm for 5 minutes. Thesupernatant from each well (100 μL) was transferred into thecorresponding wells of a flat-bottom plate and OD was read in an ELISAreader at 415-490 nm. Results are reported as the ratio of the OD at 415nm to that at 490 nm.

Results:

FIG. 23 graphically illustrates hemolysis (as measured by hemoglobinrelease of lysed rabbit erythrocytes into the supernatant measured byphotometry) of mannan-coated rabbit erythrocytes by mouse serum over arange of serum concentrations in serum from MASP-1/3−/− and WT control.As shown in FIG. 23, it is demonstrated that inhibiting MASP-3 preventscomplement-mediated lysis of mannan-coated WT rabbit erythrocytes. Theseresults further support the use of MASP-3 inhibitors for the treatmentof one or more aspects of PNH as described in Example 5.

Example 7

This Example describes the generation of MASP-1 and MASP-3 monoclonalantibodies using an in vitro system comprising a modified DT40 cellline, DTLacO.

Background/Rationale:

Antibodies against human MASP-1 and MASP-3 were generated using an invitro system comprising a modified DT40 cell line, DTLacO, that permitsreversible induction of diversification of a particular polypeptide, asfurther described in WO2009029315 and US2010093033. DT40 is a chicken Bcell line that is known to constitutively mutate its heavy and lightchain immunoglobulin (Ig) genes in culture. Like other B cells, thisconstitutive mutagenesis targets mutations to the V region of Ig genes,and thus, the CDRs of the expressed antibody molecules. Constitutivemutagenesis in DT40 cells takes place by gene conversion using as donorsequences an array of non-functional V gene segments (pseudo-V genes;ψV) situated upstream of each functional V region. Deletion of the ψVregion was previously shown to cause a switch in the mechanism ofdiversification from gene conversion to somatic hypermutation, themechanism commonly observed in human B cells. The DT40 chicken B celllymphoma line has been shown to be a promising starting point forantibody evolution ex vivo (Cumbers, S. J. et al. Nat Biotechnol 20,1129-1134 (2002); Seo, H. et al. Nat Biotechnol 23, 731-735 (2005)).DT40 cells proliferate robustly in culture, with an 8-10 hour doublingtime (compared to 20-24 hr for human B cell lines), and they supportvery efficient homologous gene targeting (Buerstedde, J. M. et al. EmboJ 9, 921-927 (1990)). DT40 cells command enormous potential V regionsequence diversity given that they can access two distinct physiologicalpathways for diversification, gene conversion and somatic hypermutation,which create templated and nontemplated mutations, respectively(Maizels, N. Annu Rev Genet 39, 23-46 (2005)). Diversified heavy andlight chain immunoglobulins (Igs) are expressed in the form of acell-surface displayed IgM. Surface IgM has a bivalent form,structurally similar to an IgG molecule. Cells that display IgM withspecificity for a particular antigen can be isolated by binding eitherimmobilized soluble or membrane displayed versions of the antigen.However, utility of DT40 cells for antibody evolution has been limitedin practice because—as in other transformed B cell lines—diversificationoccurs at less than 1% the physiological rate.

In the system used in this example, as described in WO2009029315 andUS2010093033, the DT40 cells were engineered to accelerate the rate ofIg gene diversification without sacrificing the capacity for furthergenetic modification or the potential for both gene conversion andsomatic hypermutation to contribute to mutagenesis. Two keymodifications to DT40 were made to increase the rate of diversificationand, consequently, the complexity of binding specificities in ourlibrary of cells. First, Ig gene diversification was put under thecontrol of the potent E. coli lactose operator/repressor regulatorynetwork. Multimers consisting of approximately 100 polymerized repeatsof the potent E. coli lactose operator (PolyLacO) were inserted upstreamof the rearranged and expressed Igλ, and IgH genes by homologous genetargeting. Regulatory factors fused to lactose repressor protein (Lad)can then be tethered to the LacO regulatory elements to regulatediversification, taking advantage of the high affinity (k_(D)=10⁻¹⁴ M)of lactose repressor for operator DNA. DT40 PolyLacO-λ_(R) cells, inwhich PolyLacO was integrated only at Igλ, exhibited a 5-fold increasein Ig gene diversification rate relative to the parental DT40 cellsprior to any engineering (Cummings, W. J. et al. PLoS Biol 5, e246(2007)). Diversification was further elevated in cells engineered tocarry PolyLacO targeted to both the IgX, and the IgH genes (“DTLacO”).DTLacO cells were demonstrated to have diversification rates 2.5- to9.2-fold elevated relative to the 2.8% characteristic of the parentalDT40 PolyLacO-λ_(R) LacI-HP1 line. Thus, targeting PolyLacO elements toboth the heavy and light chain genes accelerated diversification21.7-fold relative to the DT40 parental cell line. Tethering regulatoryfactors to the Ig loci not only alters the frequency of mutagenesis, butalso can change the pathway of mutagenesis creating a larger collectionof unique sequence changes (Cummings et al. 2007; Cummings et al. 2008).Second, a diverse collection of sequence starting points for thetethered factor-accelerated Ig gene diversification was generated. Thesediverse sequence starting points were added to DTLacO by targetingrearranged Ig heavy-chain variable regions, isolated from a two monthold chick, to the heavy chain locus. The addition of these heavy chainvariable regions created a repertoire of 10′ new starting points forantibody diversification. Building these new starting points into theDTLacO cell line permits the identification of clones that bind aparticular target, and then rapid affinity maturation by the tetheredfactors. Following affinity maturation, a full-length, recombinantchimeric IgG is made by cloning the matured, rearranged heavy- andlight-chain variable sequences (VH and Vλ; consisting of chickenframework regions and the complementarity determining regions or CDRs)into expression vectors containing human IgG1 and lambda constantregions. These recombinant mAbs are suitable for in vitro and in vivoapplications, and they serve as the starting point for humanization.

Methods:

Selection for MASP-1 and MASP-3 Antigen Binding.

Initial selections were performed by binding DTLacO populationsdiversified by gene targeting to beads complexed with human MASP-1 (SEQID NO:8) and MASP-3 antigen (SEQ ID NO:2); and subsequent selections byFACS, using fluorescence-labeled soluble antigen (Cumbers, S. J. et al.Nat Biotechnol 20, 1129-1134 (2002); Seo, H. et al. Nat Biotechnol 23,731-735 (2005). Because of the conserved amino acid sequence in thealpha chain that is shared between MASP-1 and MASP-3 (shown in FIG. 2),and the distinct beta chain sequences (shown in FIG. 2), separate,parallel screens for binders to MASP-1 and MASP-3 were carried out toidentify MASP-1 specific mAbs, MASP-3 specific mAbs and also mAbscapable of binding to both MASP-1 and MASP-3 (dual-specific). Two formsof antigen were used to select and screen for binders. First,recombinant MASP-1 or MASP-3, either full-length or a fragment, fused toan Fc domain were bound to Dynal magnetic Protein G beads or used inFACS-based selections using a PECy5-labeled anti-human IgG(Fc) secondaryantibody. Alternatively, recombinant versions of MASP-1 or MASP-3proteins were directly labeled with Dylight flours and used forselections and screening.

Binding and Affinity.

Recombinant antibodies were generated by cloning PCR-amplified V regionsinto a vector that supported expression of human IgG1 in 293F cells(Yabuki et al., PLoS ONE, 7(4):e36032 (2012)). Saturation bindingkinetics were determined by staining DTLacO cells expressing antibodybinding MASP-1 or MASP-3 with various concentrations offluorescent-labeled soluble antigen. Functional assays for MASP-3specific activity including MASP-3-dependent C3b deposition andMASP-3-dependent factor D cleavage were carried out as described inExamples 8 and 9, respectively. A functional assay for MASP-1-specificactivity, namely the inhibition of MASP-1-dependent C3b deposition wascarried out as described below.

Results:

Numerous MASP-1 and MASP-3 binding antibodies were generated using themethods described above. Binding, as demonstrated by FACS analysis, isdescribed for the representative clones M3J5 and M3M1, which wereisolated in screens for MASP-3 binders.

FIG. 24A is a FACS histogram of MASP-3 antigen/antibody binding forDTLacO clone M3J5. FIG. 24B is a FACS histogram of MASP-3antigen/antibody binding for DTLacO clone M3M1. In FIGS. 24A and 24B thegray filled curves are IgG1-stained negative control, and thick blackcurves are MASP-3-staining.

FIG. 25 graphically illustrates a saturation binding curve of clone M3J5(Clone 5) for the MASP-3 antigen. As shown in FIG. 25, the apparentbinding affinity of the M3J5 antibody for MASP-3 is about 31 nM.

Sequence analysis of identified clones was performed using standardmethods. All clones were compared to the common (DT40) VH and VLsequences and to each other. Sequences for the two afore-mentionedclones, M3J5 and M3M1 are provided in an alignment with two additionalrepresentative clones, D14 and 1E10, which were identified in screensfor CCP1-CCP2-SP fragments of MASP-1 and MASP-3, respectively. D14 and1E10 bind regions common to both MASP-1 and MASP-3.

FIG. 26A is an amino acid sequence alignment of the VH regions of M3J5,M3M1, D14 and 1E10 to the chicken DT40 VH sequence.

FIG. 26B is an amino acid sequence alignment of the VL regions of M3J5,M3M1, D14 and 1E10 to the chicken DT40 VL sequence.

The VH and VL amino acid sequence of each clone is provided below.

Heavy Chain Variable Region (VII) Sequences

FIG. 26A shows an amino acid alignment of the heavy-Chain VariableRegion (VH) sequences for the parent DTLacO (SEQ ID NO:300), theMASP-3-binding clones M3J5 (SEQ ID NO:301), and M3M1 (SEQ ID NO:302),and the MASP-1/MASP-3 dual binding clones D14 (SEQ ID NO:306), and 1E10(SEQ ID NO:308).

The Kabat CDRs in the VH sequences below are located at the followingamino acid positions: H1:aa 31-35; H2:aa 50-62; and H3:aa 95-102.

The Chothia CDRs in the VH sequences below are located at the followingamino acid positions: H1: aa 26-32; H2: aa 52-56; and H3: aa 95-101.

Parent DTLacO VH:  (SEQ ID NO: 300)AVTLDESGGGLQTPGGALSLVCKASGFTFSSNAMGWVRQAPGKGLEWVAGIDDDGSGTRYAPAVKGRATISRDNGQSTLRLQLNNLRAEDTGTYYCTKCAYSSGCDYEGGYIDAWGHGTEVIVSS Clone M3J5 VH:  (SEQ ID NO: 301)AVTLDESGGGLQTPGGGLSLVCKASGFTFSSYAMGWMRQAPGKGLEYVAGIRSDGSFTLYATAVKGRATISRDNGQSTVRLQLNNLRAEDTATYFCTRSG NVGDIDAWGHGTEVIVSSClone M3M1 VH:  (SEQ ID NO: 302)AVTLDESGGGLQTPGGGLSLVCKASGFDFSSYQMNWIRQAPGKGLEFVAAINRFGNSTGHGAAVKGRVTISRDDGQSTVRLQLSNLRAEDTATYYCAKGVYGYCGSYSCCGVDTIDAWGHGTEVIVSS Clone D14 VH:  (SEQ ID NO: 306)AVTLDESGGGLQTPGGALSLVCKASGFTFSSYAMHWVRQAPGKGLEWVAGIYKSGAGTNYAPAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKTTGSGCSSGYRAEYIDAWGHGTEVIVSS Clone 1E10 VH:  (SEQ ID NO: 308)AVTLDESGGGLQTPGGALSLVCKASGFTFSSYDMVWVRQAPGKGLEFVAGISRNDGRYTEYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTATYYCARDAGGSAYWFDAGQIDAWGHGTEVIVSS

Light Chain Variable Region (VL) sequences

FIG. 26B shows an amino acid alignment of the light-Chain VariableRegion (VL) sequences for the parent DTLacO (SEQ ID NO:303) and theMASP-3-binding clones M3J5 (SEQ ID NO:304), and M3M1 (SEQ ID NO:305),and the MASP-1/MASP-3 dual binding clones D14 (SEQ ID NO:307) and 1E10(SEQ ID NO:309).

Parent DTLacO VL:  (SEQ ID NO: 303)ALTQPASVSANLGGTVKITCSGGGSYAGSYYYGWYQQKSPGSAPVTVIYDNDKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGA AFGAGTTLTVLClone M3J5 VL:  (SEQ ID NO: 304)ALTQPASVSANPGETVKITCSGGYSGYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSG AAFGAGTTLTVLClone M3M1 VL:  (SEQ ID NO: 305)ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGA AFGAGTTLTVLClone D14 VL: (SEQ ID NO: 307)ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGA AFGAGTTLTVLClone 1E10 VL:  (SEQ ID NO: 309)ALTQPASVSANPGETVKITCSGGGSYAGSYYYGWYQQKAPGSAPVTLIYYNNKRPSDIPSRFSGSLSGSTNTLTITGVRADDEAVYFCGSADNSGA AFGAGTTLTVL

LEA-2 (MASP-2-dependent) Functional Assay

MASP-1 contributes to LEA-2 via its ability to activate MASP-2 (see FIG.1). The Wieslab® Complement System Screen MBL assay (Euro Diagnostica,Malmo, Sweden) measures C5b-C9 deposition under conditions that isolateLEA-2-dependent activation (i.e., traditional lectin pathway activity).The assay was carried out according to the manufacturer's instructionswith representative clone 1E10 tested as a final concentration of 400nM.

FIG. 27 is a bar graph showing the inhibitory activity of the mAb 1E10in comparison to the positive serum provided with the assay kit, as wellas an isotype control antibody. As shown in FIG. 27, mAb 1E10demonstrates partial inhibition of LEA-2-dependent activation (viainhibition of MASP-1-dependent activation of MASP-2), whereas theisotype control antibody does not. Stronger inhibition should beachieved by continued affinity maturation of this antibody for MASP-1binding using the tethered factors in the DTLacO system.

LEA-1 (MASP-3-dependent) Function Assays for representative mAbs aredescribed below in Examples 8 and 9.

Summary of Results:

The above results showed that the DTLacO platform permitted rapid exvivo discovery of MASP-1 and MASP-3 monoclonal antibodies withinhibitory properties on LEA-1 (as shown below in Examples 8 and 9) andon LEA-2 (as shown in this Example).

Example 8

Analysis of the complement pathway in 3MC serum with S. aureus

Background/Rationale:

It was determined that MASP-3 is not activated through exposure tonon-immobilized fluid-phase mannan, zymosan A or N-acetyl cysteineeither in the presence or absence of normal human serum. However, it wasdetermined that recombinant and native MASP-3 are activated on thesurface of heat-inactivated S. aureus in the presence and absence ofnormal human serum (NETS) or heat-inactivated human serum (HIS) (datanot shown). It was also determined that C3b deposition occurs on thesurface of S. aureus in the presence of normal human serum, and that thedeposition can be monitored using a flow cytometer. Therefore, thealternative pathway (AP) response to S. aureus was measured as describedin this Example as a means of assessing the contribution of MASP-3 toLEA-1.

Methods:

Recombinant MASP-3: polynucleotide sequences encoding full lengthrecombinant human MASP-3, a truncated serine protease (SP) activeversion of MASP-3 (CCP1-CCP2-SP), and a SP-inactivated form of MASP-3(S679A) were cloned into the pTriEx7 mammalian expression vector(Invivogen). The resulting expression constructs encode the full lengthMASP-3 or the CCP1-CCP2-SP fragment with an amino-terminal Streptag anda carboxy-terminal His6 tag. The expression constructs were transfectedinto Freestyle 293-F or Expi293F cells (Invitrogen) according to theprotocols provided by the manufacturer. After three to four days ofculture in 5% CO2 at 37° C., recombinant proteins were purifiedutilizing Streptactin affinity chromatography.

Recombinant MASP-1: the full length or truncated CCP1-CCP2-SP forms ofrecombinant MASP-1 with or without the stabilizing R504Q (Dobo et al.,J. Immunol 183:1207, 2009) or SP inactivating (S646A) mutations andbearing an amino-terminal Steptag and a carboxy-terminal His6 tag weregenerated as described for recombinant MASP-3 above.

1. C3b Deposition and Factor B Cleavage on S. aureus in 3MC (Human)Serum

An initial experiment was carried out to demonstrate that the flowcytometry assay is able to detect the presence or absence of AP-drivenC3b deposition (AP-C3b) as follows. Five percent of the following sera:normal human serum, factor B (Factor B)- depleted human serum, factorD-depleted human serum and properdin-depleted human serum (obtained fromComplement Technology, Tyler, Tex., USA) were mixed with test antibodyin either Mg⁺⁺/EGTA buffer or EDTA at 4° C. overnight. Heat-killed S.aureus (10⁸/reaction) was added to each mixture to a total volume of 100μL and rotated at 37° C. for 40 minutes. Bacteria were washed in washingbuffer, the bacterial pellet was re-suspended in washing buffer and a 80μL aliquot of each sample was analyzed for C3b deposition on thebacterial surface, which was detected with anti-human C3c (Dako, UK)using flow cytometry.

The results of the flow cytometry detection of C3b are shown in FIG.28A. As shown in FIG. 28A, panel 1, normal human serum in the presenceof EDTA, which is known to inactivate the AP, no C3b deposition wasobserved (negative control). In normal human serum treated withMg⁺⁺/EGTA, only lectin-independent complement pathways can function. Inpanel 2, Mg⁺⁻/EGTA buffer is used, therefore the AP is active, andAP-driven C3b deposition is observed (positive control). As shown inpanel 3, 4 and 5, in factor B-depleted, factor D-depleted andproperdin-depleted serum, respectively, no alternative pathway drivenC3b deposition is observed, as expected. These results demonstrate thatthe assay is capable of detecting AP-dependent C3b deposition.

A C3b deposition on S. aureus assay was carried out as described aboveto assess the ability of recombinant MASP-3 to reconstitute the AP(LEA-1) in human 3MC serum, which is deficient in MASP-3 (Rooryck C, etal., Nat Genet. 43(3):197-203 (2011)). The following combinations ofreagents were tested.

-   1. 5% normal human serum+EDTA-   2. 5% normal human serum+Mg/EGTA-   3. 5% human 3MC (MASP-3^(−/−)) serum+Mg^(+/+)/EGTA-   4. 5% human 3MC (MASP-3^(−/−)) serum+Mg⁺⁺/EGTA plus active    full-length rMASP-3-   5. 5% human 3MC (MASP-3^(−/−)) serum+Mg⁺⁺/EGTA plus truncated active    rMASP-3 (CCP1/CCP2/SP)-   6. 5% human 3MC (MASP-3^(−/−)) serum+Mg⁺⁻/EGTA plus inactive rMASP-3    (S679A)-   7. 5% human 3MC (MASP-3^(−/−)) serum+Mg⁺⁻/EGTA plus active full    length rMASP-1.

The various mixtures of 5% serum and recombinant proteins (5 μg of each)as shown above were incubated in the specified buffer conditions (eitherMg⁺⁺ /EGTA buffer or EDTA) at 4° C. overnight. After the incubationovernight, 10⁸ heat-killed S. aureus were added to each mixture in atotal volume of 100 μL and rotated at 37° C. for 40 minutes. Bacteriawere washed and re-suspended in washing buffer and an 80 μL aliquot ofeach sample was analyzed for C3b deposition by FACS. The remaining 20 μLaliquot of each sample was used to measure factor B cleavage by Westernblot using anti-factor B antibody as described below.

The results of the flow cytometery detection of C3b are shown in FIG.28B. Panel numbers correspond to the numbers designated for each of thereagent combination outlined above. The negative control (panel 1) andpositive control (panel 2) show the absence and presence of C3bdeposition, as expected. Panel 3 shows that AP-driven C3b deposition isabsent in 3MC serum. Panels 4 and 5 show that active full length rMASP-3(panel 4) and active rMASP-3 (CCP1-CCP2-SP) (panel 5) both restoreAP-driven C3b deposition in 3MC serum. Panel 6 shows that inactiverMASP-3 (S679A) does not restore AP-driven C3b deposition in 3MC serum.Panel 7 shows that rMASP-1 does not restore AP-driven C3b deposition in3MC serum.

Taken together, these results demonstrate that MASP-3 is required forAP-driven C3b deposition on S. aureus in human serum.

MASP-3-Dependent Activation of Factor B

In order to analyze MASP-3-dependent activation of Factor B, the variousmixtures of 5% serum (either normal human serum or 3MC patient serum)and recombinant proteins as shown above were assayed as described above.From each reaction mixture, 20 μL were removed and added to proteinsample loading buffer. The samples were heated at 70° C. for 10 minutesand loaded onto an SDS-PAGE gel. Western blot analysis was performedusing a Factor B polyclonal antibody (R&D Systems). Activation of FactorB was apparent by the formation of two lower molecular weight cleavageproducts (Bb and Ba) derived from the higher molecular weight pro-FactorB protein.

FIG. 29 shows the results of a Western blot analysis to determine factorB cleavage in response to S. aureus in 3MC serum in the presence orabsence of rMASP-3. As shown in lane 1, the normal human serum in thepresence of EDTA (negative control) demonstrates very little Factor Bcleavage relative to normal human serum in the presence of Mg⁺⁺/EGTA,shown in lane 2 (positive control). As shown in lane 3, 3MC serumdemonstrates very little Factor B cleavage in the presence of Mg⁺⁺/EGTA.However, as shown in lane 4, Factor B cleavage is restored by theaddition and pre-incubation of full-length, recombinant MASP-3 protein(5 μg) to the 3MC serum.

Assay to Determine the Effect of rMASP-3 on Pro-Factor D in FactorB/C3(H₂O) Cleavage

The following assay was carried out to determine the minimal requirementfor MASP-3-dependent activation/cleavage of factor B.

C3(H₂O) (200 ng), purified plasm factor B (20 μg), recombinantpro-factor D (200 ng) and recombinant human MASP-3 (200 ng) were mixedtogether in various combinations (as shown in FIG. 30), in a totalvolume of 100 μL in BBS/Ca⁺⁻/Mg⁺⁺ and incubated at 30° C. for 30minutes. The reaction was stopped by adding 25 uL of SDS loading dyecontaining 5% 2-mercaptoethanol. After boiling at 95° C. for 10 minutesunder shaking (300 rpm), the mixture was spun down at 1400 rpm for 5minutes and 20 uL of the supernatant was loaded and separated on a 10%SDS gel. The gel was stained with Coomassie brilliant blue.

Results:

FIG. 30 shows a Comassie-stained SDS-PAGE gel in which factor B cleavageis analyzed. As shown in lane 1, factor B cleavage is most optimal inthe presence of C3, MASP-3 and pro-factor D. As shown in lane 2, C3 isabsolutely required; however, as shown in lanes 4 and 5, either MASP-3or pro-factor D are able to mediate factor B cleavage, as long as C3 ispresent.

Analysis of the Ability of MASP-3 mAbs to Inhibit MASP-3-DependentAP-Driven C3b Deposition

As described in this Example it was demonstrated that MASP-3 is requiredfor AP-driven C3b deposition on S. aureus in human serum. Therefore, thefollowing assay was carried out to determine if a representative MASP-3mAb identified as described in Example 7, could inhibit activity ofMASP-3. Active, recombinant MASP-3 (CCP1-CCP2-SP) fragment protein (250ng) was pre-incubated with an isotype control mAb, mAb1A5 (controlobtained from the DTLacO platform that does not bind MASP-3 or MASP-1),or mAbD14 (binds MASP-3) at three different concentrations (0.5, 2 and 4μM) for 1 hour on ice. The enzyme-mAb mixture was exposed to 5% 3MCserum (MASP-3 deficient) and 5×10⁷ heat-killed S. aureus in a finalreaction volume of 50 μL. The reactions were incubated at 37° C. for 30minutes, and then stained for the detection of C3b deposition. Thestained bacterial cells were analyzed by a flow cytometer.

FIG. 31 graphically illustrates the mean fluorescent intensities (MFI)of C3b staining obtained from the three antibodies plotted as a functionof mAb concentration in 3MC serum with the presence of rMASP-3. As shownin FIG. 31, mAbD14 demonstrates inhibition of C3b deposition in aconcentration-dependent manner. In contrast, neither of the control mAbsinhibited C3b deposition. These results demonstrate that mAbD14 is ableto inhibit MASP-3-dependent C3b deposition. Improved inhibitory activityfor mAbD14 is expected following continued affinity maturation of thisantibody for MASP-3 binding using the tethered factors in the DTLacOsystem.

Summary of Results:

In summary, the results in this Example demonstrate a clear defect ofthe AP in serum deficient for MASP-3. Thus, MASP-3 has been demonstratedto make a critical contribution to the AP, using factor B activation andC3b deposition as functional end-points. Furthermore, addition offunctional, recombinant MASP-3, including the catalytically-activeC-terminal portion of MASP-3 corrects the defect in factor B activationand C3b deposition in the serum from the 3MC patient. Conversely, asfurther demonstrated in this Example, addition of a MASP-3 antibody(e.g., mAbD14) in 3MC serum with rMASP-3 inhibits AP-driven C3bdeposition. A direct role of MASP-3 in Factor B activation, andtherefore the AP, is demonstrated by the observation that recombinantMASP-3, along with C3, is sufficient to activate recombinant factor B.

Example 9

This Example demonstrates that MASP-1 and MASP-3 activate factor D.

Methods:

Recombinant MASP-1 and MASP-3 were tested for their ability to cleavetwo different recombinant versions of pro-factor D. The first version(pro-factor D-His) lacks an N-terminal tag, but has a C-terminal Histag. Thus, this version of pro-factor D contains the 5 amino acidpro-peptide that is removed by cleavage during activation. The secondversion (ST-pro-factor D-His) has a Strep-TagII sequence on theN-terminus, thus increasing the cleaved N-terminal fragment to 15 aminoacids. ST-pro-factor D also contains a His6 tag at the C-terminus. Theincreased length of the propeptide of ST-pro-factor D-His improves theresolution between the cleaved and uncleaved forms by SDS-PAGE comparedto the resolution possible with the pro-factor D-HIS form.

Recombinant MASP-1 or MASP-3 proteins (2 ug) was added to eitherpro-factor D-His or ST-pro-factor D-His substrates (100 ng) andincubated for 1 hour at 37° C. The reactions were electrophoresed on a12% Bis-Tris gel to resolve pro-factor D and the active factor Dcleavage product. The resolved proteins were transferred to a PVDFmembrane and analyzed by Western blot by detection with a biotinylatedfactor D antibody (R&D Systems).

Results:

FIG. 32 shows the Western blot analysis of pro-factor D substratecleavage.

TABLE 14 Lane Description for Western Blot shown in FIG. 32 Experimentalconditions Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Pro-Factor D + + + + +rMASP-3 − + − − − (full-length) rMASP-3a − − + − − (S679A) rMASP-1A − −− + − (S646A) rMASP-1 − − − − + (CCP-1- CCP2-SP)

As shown in FIG. 32, only full length MASP-3 (lane 2) and the MASP-1CCP1-CCP2-SP) fragment (lane 5) cleaved ST-pro-factor D-His₆. Thecatalytically-inactive full length MASP-3 (S679A; lane 3) and MASP-1(S646A; lane 3) failed to cleave either substrate. Identical resultswere obtained with the pro-factor D-His6 polypeptide (not shown). Thecomparison of a molar excess of MASP-1 (CCP1-CCP2-SP) relative to MASP-3suggests that MASP-3 is a more effective catalyst of pro-factor Dcleavage than is MASP-1, as least under the condtions described herein.

Conclusions: Both MASP-1 and MASP-3 are capable of cleaving andactivating factor D. This activity directly connects LEA-1 with theactivation of the AP. More specifically, activation of factor D byMASP-1 or MASP-3 will lead to factor B activation, C3b deposition, andlikely opsonization and/or lysis.

Assay for Inhibition of MASP-3-Dependent Cleavage of Pro-Factor D withMASP-3 Antibodies

An assay was carried out to determine the inhibitory effect ofrepresentative MASP-3 and MASP-1 mAbs, identified as described inExample 7, on MASP-3-dependent factor D cleavage as follows. Active,recombinant MASP-3 protein (80 ng) was pre-incubated with 1μg ofrepresentative mAbs D14, M3M1 and a control antibody (which bindsspecifically to MASP-1, but not to MASP-3) at room temperature for 15minutes. Pro-factor D with an N-terminal Strep-tag (ST-pro-factor D-His,70 ng) was added and the mixture was incubated at 37° C. for 75 minutes.The reactions were then electrophoresed, blotted and stained withanti-factor D as described above.

FIG. 33 is a Western blot showing the partial inhibitory activity of themAbs D14 and M3M1 in comparison to a control reaction containing onlyMASP-3 and ST-pro-factor D-His (no mAb; lane 1), as well as a controlreaction containing a mAb obtained from the DTLacO library that bindsMASP-1, but not MASP-3 (lane 4). As shown in FIG. 33, in the absence ofan inhibitory antibody, MASP-3 cleaves approximately 50% of pro-factor Dinto factor D (lane 1). The control MASP-1 specific antibody (lane 4)does not change the ratio of pro-factor D to factor D. In contrast, asshown in lanes 2 and 3, both mAb D14 and mAb M3M1 inhibitMASP-3-dependent cleavage of pro-factor D to factor D, resulting in areduction in factor D generated.

Conclusions: These results demonstrate that MASP-3 mAbs D14 and M3M1 areable to inhibit MASP-3-dependent factor D cleavage. Improved inhibitoryactivity for mAbD14 and mAb M3M1 is expected following continuedaffinity maturation of these antibodies for MASP-3 binding using thetethered factors in the DTLacO system.

Example 10

This Example demonstrates that MASP-3 deficiency preventscomplement-mediated lysis of mannan-coated WT rabbit erythrocytes.

Background/Rationale:

As described in Examples 5 and 6 herein, the effect of MASP-2- andMASP-3-deficient serum on lysis of red blood cells from blood samplesobtained from a mouse model of PNH demonstrated the efficacy of MASP-2inhibition and/or MASP-3 inhibition to treat subjects suffering fromPNH, and also supported the use of inhibitors of MASP-2 and/orinhibitors of MASP-3 (including dual or bi-specific MASP-2/MASP-3inhibitors) to ameliorate the effects of C3 fragment-mediatedextravascular hemolysis in PNH subjects undergoing therapy with a C5inhibitor such as eculizumab.

As described in this Example, C3b deposition experiments and hemolysisexperiments were carried out in MASP-3 deficient serum from additional3MC patients, confirming the results obtained in Examples 5 and 6. Inaddition, experiments were carried out which demonstrated that additionof rMASP-3 to 3MC serum was able to reconstitute C3b deposition andhemolytic activity.

Methods:

MASP-3-deficient serum was obtained from three different 3MC patients asfollows:

-   3MC Patient 1: contains an allele bearing a mutation that renders    the exon encoding the MASP-3 serine protease domain dysfunctional,    supplied along with the mother and father of the 3MC patient (both    heterozygous for the allele bearing a mutation that renders the exon    encoding the MASP-3 serine protease domain dysfunctional),-   3MC Patient 2: Has C1489T (H497Y) mutation in exon 12 of MASP-1, the    exon that encodes the serine protease domain of MASP-3, resulting in    nonfunctional MASP-3, but functional MASP-1 proteins.-   3MC Patient 3: Has a confirmed defect in the MASP-1 gene, resulting    in nonfunctional MASP-3 and nonfunctional MASP-1 proteins.

Experiment #1: C3b Deposition Assay

An AP assay was carried out under traditional AP-specific conditions(BBS/Mg⁺⁺/EGTA, without Ca⁺⁺, wherein BBS=barbital buffered salinecontaining sucrose), as described in Bitter-Suermann et al., Eur. J.Immunol 11:291-295 (1981)), on zymosan-coated microtiter plates at serumconcentrations ranging from 0.5 to 25% and C3b deposition was measuredover time.

Results:

FIG. 34 graphically illustrates the level of AP-driven C3b deposition onzymosan-coated microtiter plates as a function of serum concentration inserum samples obtained from MASP-3-deficient (3MC), C4-deficient andMBL-deficient subjects. As shown in FIG. 34, and summarized below inTABLE 15, MASP-3-deficient patient sera from Patient 2 and Patient 3have residual AP activity at high concentrations (25%, 12.5%, 6.25%serum concentrations), but a significantly higher AP₅₀ (i.e., 8.2% and12.3% of serum needed to achieve 50% of maximum C3 deposition).

FIG. 35A graphically illustrates the level of AP-driven C3b depositionon zymosan-coated microtiter plates under “traditional” AP-specificconditions (i.e., BBS/EGTA/Mg⁻⁺ without Ca⁺⁺) as a function of time in10% human serum samples obtained from MASP-3 deficient, C4-deficient andMBL-deficient human subjects.

TABLE 15 below summarizes the AP₅₀ results shown in FIG. 34 and thehalf-times for C3b deposition shown in FIG. 35A.

TABLE 15 Summary of Results shown in FIGS. 34 and 35A Serum type AP₅₀(%) T_(1/2) (min) Normal 4.5 26.3 MBL-deficient (MBL−/−) 5.7 27.5C4-deficient (C4−/−) 5.1 28.6 3MC (Patient 3) 8.2 58.2 3MC (Patient 2)12.3 72.4 Note: In BBS/Mg⁺⁺/EGTA buffer, the lectin pathway-mediatedeffects are deficient due to absence of Ca⁺⁺ in this buffer.

Experiment #2: Analysis of Pro-Factor D Cleavage in 3MC Patient Sera byWestern Blot

Methods: Serum was obtained from 3MC patient #2 (MASP-3 (−/−), MASP-1(+/+) and from 3MC patient #3 (MASP-3 (−/−), MASP-1 (−/−). The patientsera, along with sera from normal donors (W), were separated bySDS-polyacrylamide gel and the resolved proteins were blotted to apolyvinylidine fluoride membrane. Human pro-factor D (25,040 Da) and/ormature factor D (24,405 Da) were detected with a human factor D-specificantibody.

Results: The results of the Western blot are shown in FIG. 35B. As shownin FIG. 35B, in the sera from normal donors (W), the factor D antibodydetected a protein of a size consistent with mature factor D (24,405Da). As further shown in FIG. 35B, the factor D antibody detected aslightly larger protein in the sera from 3MC patient #2 (P2) and 3MCpatient #3 (P3), consistent with the presence of pro-factor D (25,040Da) in these 3MC patients.

Experiment #3: Wieslab Complement Assays with 3MC Patient Sera

Methods: Sera obtained from 3MC patient #2 (MASP-3 (−/−), MASP-1 (+/+))and from 3MC patient #3 (MASP-3 (−/−), MASP-1 (−/−)) were also testedfor classical, lectin and alternative pathway activity using the WieslabComplement System Screen (Euro-Diagnostica, Malmo, Sweden) according tothe manufacturer's instructions. Normal human serum was tested inparallel as a control.

Results: FIG. 35C graphically illustrates the results of the Weislabclassical, lectin and alternative pathway assays with plasma obtainedfrom 3MC patient #2, 3MC patient #3, and normal human serum. As shown inFIG. 35C, under conditions of the Wieslab assay, the classical,alternative, and MBL (lectin) pathways are all functional in the normalhuman serum. In serum from 3MC patient #2 (MASP-3 (−/−), MASP-1 (+/+)),the classical pathway and lectin pathway are functional, however thereis no detectable alternative pathway activity. In serum from 3MC patient#3 (MASP-3 (−/−), MASP-1 (−/−)), the classical pathway is functional,however there is no detectable lectin pathway activity and no detectablealternative pathway activity.

The result in FIGS. 35B and 35C further support our understanding of therole of MASP-1 and MASP-3 in the LEA-1 and LEA-2 pathways. Specifically,the absence of the alternative pathway with a nearly fully functionallectin pathway in serum from Patient 2, who lacks only MASP-3, confirmsthat MASP-3 is essential for activation of the alternative pathway.Serum from Patient 3, who lacks both MASP-1 and MASP-3, has lost theability to activate the lectin pathway as well as the alternativepathway. This result confirms the requirement of MASP-1 for a functionalLEA-2 pathway, and is consistent with Example 7, and the literaturedemonstrating that MASP-1 activates MASP-2. The apparent inability ofboth sera to activate pro-factor D is also consistent with the datadescribed in Example 9 demonstrating that MASP-3 cleaves pro-factor D.These observations are consistent with the LEA-1 and LEA-2 pathways asdiagrammed in FIG. 1.

Experiment #4: Hemolysis Assay Testing Mannan-Coated Rabbit Erythrocytesfor Lysis in the presence of human normal or 3MC serum (in the absenceof Ca⁺⁺)

Methods:

Preparation of Rabbit RBC in the Absence of Ca' (i.e., by using EGTA)

Rabbit whole blood (2 mL) was split into two 1.5 mL eppendorf tubes andcentrifuged for 3 minutes at 8000 rpm (approximately 5.9 rcf) in arefrigerated eppendorf centrifuge at 4° C. The RBC pellet was washedthree times after re-suspending in ice-cold BBS/ Mg⁺⁺/Ca⁺⁺ (4.4 mMbarbituric acid, 1.8 mM sodium barbitone, 145 mM NaCl, pH 7.4, 5 mM 5 mMCa⁺⁺). After the third wash, the pellet was re-suspended in 4 mL BBS/Mg⁺⁺/Ca⁺⁺. The erythrocytes were pelleted and the RBCs were washed withBBS/0.1% gelatin/Mg⁺⁺/Ca⁺⁺ as described above. The RBCs suspension wasstored in BBS/0.1% gelatin/ Mg⁺⁺/Ca⁺⁺ at 4° C. Then, 100 μL of suspendedRBCs were diluted with 1.4 mL water and spun down at 8000 rpm(approximately 5.9 rcf) for 3 minutes and the OD of the supernatant wasadjusted to 0.7 at 541 nm (an OD of 0.7 at 541 nm corresponds toapproximately 10⁹ erythrocytes/nil). After that, 1 mL of the resuspendedRBCs at OD 0.7 were added to 9 ml of BBS/Mg⁺⁺/EGTA in order to achieve aconcentration of 10⁸ erythrocytes/ml. Dilutions of the test sera orplasma were prepared in ice-cold BBS, Mg⁺⁺, EGTA and 100 μL of eachserum or plasma dilution was pipetted into the corresponding well ofround-bottom plate. 100 μL of appropriately diluted RBC (10⁸erythrocytes/nil) were added to each well. Nano-water was used toproduce the positive control (100% lysis), while a dilution withBBS/Mg⁺⁺/EGTA without serum or plasma was used as a negative control.The plate was then incubated for 1 hour at 37° C. The round bottom platewas spun down at 3750 rpm for 5 minutes. Then, 100 μL of the supernatantfrom each well was transferred into the corresponding wells of aflat-bottom plate and OD was read at 415-490 nm.

Results:

FIG. 36 graphically illustrates the percent hemolysis (as measured byhemoglobin release of lysed rabbit erythrocytes into the supernatantmeasured by photometry) of mannan-coated rabbit erythrocytes over arange of serum concentrations in serum from normal subjects and from two3MC patients (Patient 2 and Patient 3), measured in the absence of Ca⁺⁺.As shown in FIG. 36, it is demonstrated that MASP-3 deficiency reducesthe percentage of complement-mediated lysis of mannan-coatederythrocytes as compared to normal human serum. The differences betweenthe two curves from the normal human serum and the two curves from the3MC patients is significant (p=0.013, Friedman test).

TABLE 16 below summarizes the AP₅₀ results shown in FIG. 36.

TABLE 16 Summary of Results shown in FIG. 36 Serum type AP₅₀ (%) Normalhuman serum #1 7.1 Normal human serum #2 8.6 3MC Patient #2 11.9 3MCPatient #3 14.3

It is noted that when the serum samples shown in TABLE 16 were pooled,the AP₅₀ value for normal human serum=7.9 and the AP₅₀ value for 3MCserum=12.8 (p=0.031, Wilcox matched-pairs signed rank test).

Experiment #5: Reconstitution of Human 3MC Serum by Recombinant MASP-3Restores AP-Driven C3b Deposition on Zymosan Coated Plates

Methods:

An AP assay was carried out under traditional AP-specific conditions(BBS/Mg⁺⁺/EGTA, without Ca⁺⁺, wherein BBS=barbital buffered salinecontaining sucrose), as described in Bitter-Suermann et al., Eur. J.Immunol 11:291-295 (1981)), on zymosan-coated microtiter plates in thefollowing serum samples (1) 5% human serum from 3MC Patient #2 with fulllength active rMASP-3 added in at a range of 0 to 20 μg/mL; (2) 10%human serum from 3MC Patient #2 with full length active rMASP-3 added inat a range of 0 to 20 μg/mL; and (3) 5% human serum from 3MC Patient #2with inactive rMASP-3A (S679A) added in at a range of 0 to 20 μg/mL.

Results:

FIG. 37 graphically illustrates the level of AP-driven C3b deposition onzymosan-coated microtiter plates as a function of the concentration ofrMASP-3 protein added to serum samples obtained from human 3MC Patient#2 (MASP-3-deficient). As shown in FIG. 37, active recombinant MASP-3protein reconstitutes AP-driven C3b deposition on zymosan-coated platesin a concentration-dependent manner. As further shown in FIG. 37, no C3bdeposition was observed in the 3MC serum containing inactive rMASP-3(S679A).

Experiment #6: Reconstitution of Human 3MC Serum by Recombinant MASP-3Restores Hemolytic Activity in 3MC Patient Serum

Methods:

A hemolytic assay was carried out using rabbit RBC using the methodsdescribed above in Experiment #2 with the following test sera at a rangeof 0 to 12% serum: (1) normal human serum; (2) 3MC patient serum; (3)3MC patient serum plus active full length rMASP-3 (20 μg/ml); and (4)heat-inactivated human serum.

Results:

FIG. 38 graphically illustrates the percent hemolysis (as measured byhemoglobin release of lysed rabbit erythrocytes into the supernatantmeasured by photometry) of mannan-coated rabbit erythrocytes over arange of serum concentrations in (1) normal human serum; (2) 3MC patientserum; (3) 3MC patient serum plus active full length rMASP-3 (20 μg/ml);and (4) heat-inactivated human serum, measured in the absence of Ca⁺⁺.As shown in FIG. 38, the percent lysis of rabbit RBC is significantlyincreased in 3MC serum including rMASP-3 as compared to the percentlysis in 3MC serum without rMASP-3 (p=0.0006).

FIG. 39 graphically illustrates the percentage of rabbit erythrocytelysis in 7% human serum from 3MC Patient 2 and from 3MC Patient 3containing active rMASP-3 at a concentration range of 0 to 110 μg/ml inBBS/Mg⁺⁺/EGTA. As shown in FIG. 39, the percentage of rabbit RBC lysisis restored with the amount of rMASP-3 in a concentration-dependentmanner up to 100% activity.

Experiment #7: Serum of MASP-3 Deficient (3MC) Patient has FunctionalMASP-2 if MBL is Present

Methods:

A C3b deposition assay was carried out using Mannan-coated ELISA platesunder to examine whether 3MC serum is deficient in LEA-2. Citrate plasmawas diluted in BBS buffer in serial dilutions (starting at 1:80, 1:160,1: 320, 1:640, 1:1280, 1:2560) and plated on Mannan-coated plates.Deposited C3b was detected using a chicken anti-human C3b assay. LEA-2driven C3b deposition (the plasma dilutions are to high for the AP andLEA-1 to work) on Mannan-coated ELISA plates was evaluated as a functionof human serum concentration in serum from a normal human subject(NETS), from two 3MC patients (Patient 2 and Patient 3), from theparents of Patient 3 and from a MBL-deficient subject.

Results:

FIG. 40 graphically illustrates the level of LEA-2-driven (i.e.,MASP-2-driven) C3b deposition on Mannan-coated ELISA plates as afunction of the concentration of human serum diluted in BBS buffer, forserum from a normal human subject (NHS), from two 3MC patients (Patient2 and Patient 3), from the parents of Patient 3 and from a MBL-deficientsubject. These data indicate that Patient 2 is MBL sufficient. However,Patient 3 and the mother of Patient 3 are MBL deficient, and thereforetheir serum does not deposit C3b on Mannan via LEA-2. Replacement of MBLin these sera restores LEA-2 mediated C3b deposition in the serum ofPatient 3 (who is homozygous for the SNP leading to MASP-3 deficiency)and his mother (who is heterozygous for the mutant MASP-3 allele) (datanot shown). This finding demonstrates that 3MC serum is not deficient inLEA-2, but rather appears to have functional MASP-2.

Overall Summary and Conclusions:

These results demonstrate that MASP-3 deficiency in human serum resultsin loss of AP activity, as manifested in reduced C3b deposition onzymosan-coated wells and reduced rabbit erythrocyte lysis. The AP can berestored in both assays by supplementing the sera with functional,recombinant human MASP-3.

Example 11

This Example demonstrates that a chimeric mouse V region/human IgG4constant region anti-human MASP-3 monoclonal antibody (mAb M3-1, alsoreferred to as mAb 13B1) is a potent inhibitor of MASP-3-mediatedAlternative Pathway Complement (APC) Activation.

Methods:

Generation of a Chimeric Mouse V Region/Human IgG Constant RegionAnti-Human MASP-3 Monoclonal Antibody (mAb M3-1)

A murine anti-human MASP-3 inhibitory antibody (mAb M3-1) was generatedby immunizing MASP-1/3 knockout mice with the human MASP-3 CCP1-CCP2-SPdomain (aa 301-728 of SEQ ID NO:2) (see also Example 14). Brieflydescribed, splenocytes from the immunized mice were fused withP3/NS1/1-Ag4-1 and supernatants from resulting hybridoma clones werescreened for the production of antibodies that bind to human MASP-3 andfor the ability to block MASP-3-mediated cleavage of complementpro-factor D (pro-CFD) to factor D (CFD). Monoclonal antibody (mAb)variable regions were isolated by RT-PCR, sequenced and cloned intohuman IgG4 expression vectors. Chimeric monoclonal antibodies wereexpressed in transiently transfected HEK293T cells, purified and testedfor binding affinity to mouse and human MASP-3 and for the ability toinhibit MASP-3-mediated cleavage of pro-CFD to CFD.

The MASP-3 inhibitory monoclonal antibody M3-1 (13B1) comprises a heavychain variable region (VH) set forth as SEQ ID NO:30 and a light chainvariable region (VL) set forth as SEQ ID NO:45. The sequences of thevariable regions of the M3-1 monoclonal antibody are provided below:

Heavy Chain Variable Region

Presented below is the heavy chain variable region (VH) sequence for mAbM3-1. The Kabat CDRs (31-35 (H1), 50-65 (H2) and 95-102 (H3) areunderlined, which correspond to amino acid residues 31-35 (H1), 50-66(H2) and 99-102 (H3) of SEQ ID NO:30.

mAb M3-1 heavy chain variable region (VH) (SEQ ID NO: 30)QVQLKQSGAELMKPGASVKLSCKATGYTFTGKWIEWVKQRPGHGLEWIGEILPGTGSTNYNEKFKGKATFTADSSSNTAYMQLSSLTTEDSAMYYCLRSE DVWGTGTTVTVSS

Light Chain Variable Region

Presented below is the light chain variable region (VL) sequence for mAbM3-1. The Kabat CDRs (24-34 (H1), 50-56 (H2) and 89-97 (H3) areunderlined, which correspond to amino acid residues 24-40 (L1); 56-62(L2) and 95-102 (L3) of SEQ ID NO:45. These regions are the same whethernumbered by the Kabat or Chothia system.

mAb M3-1 light chain variable region (VL)  (SEQ ID NO: 45)DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC KQSYNIPTFGGGTKLEIKRmAb M3-1 VH CDRs VHCDR1:   (SEQ ID NO: 84) GKWIE VHCDR2: (SEQ ID NO: 86)EILPGTGSTNYNEKFKG  VHCDR3: (SEQ ID NO: 88) SEDV  mAb M3-1 VL CDRsVLCDR1: (SEQ ID NO: 142) KSSQSLLNSRTRKNYLA  VLCDR2: (SEQ ID NO: 144)WASTRES    VLCDR3:  (SEQ ID NO: 161) KQSYNIPT 

As shown above, MASP-3 monoclonal antibody M3-1 comprises (a) a heavychain variable region comprising (i) VHCDR1 comprising SEQ ID NO:84,(ii) VHCDR2 comprising SEQ ID NO:86 and (iii) VHCDR3 comprising SEQ IDNO:88; and (b) a light chain variable region comprising (i) VLCDR1comprising SEQ ID NO:142, (ii) VLCDR2 comprising SEQ ID NO:144 and (iii)VLCDR3 comprising SEQ ID NO:161.

Binding of mAb M3-1 to Recombinant Forms of Human and Mouse MASP-3

A monovalent Fab version of M3-1 was tested for binding to recombinant,full-length human and mouse MASP-3 protein in an ELISA experiment.Binding affinity determinations were made by coating 96-well plates withan anti-MASP-3 capture antibody that binds the protein from multiplespecies. The capture antibody has been shown to bind the CCP1-CCP2region of MASP-1 and MASP-3. Full-length versions of human and mouseprotein were immobilized on ELISA plates coated with the captureantibody, and varying concentrations of M3-1 Fab were allowed to bind tothe target protein in separate wells. Bound M3-1 was detected using ananti-kappa light chain antibody that is conjugated to HRP (NovusBiologicals NBP1-75064), and was visualized with the TMB substratereagent set (BD Biosciences 555214).

FIG. 41 graphically illustrates a representative example of a bindingexperiment that was performed with human MASP-3 in which the M3-1 Fab(also referred to as 13B1) shows an apparent binding affinity (EC₅₀) ofabout 0.117 nM to the human protein.

FIG. 42 graphically illustrates a representative example of a bindingexperiment that was performed with mouse MASP-3 in which the M3-1 Fabshows an apparent binding affinity (EC₅₀) of about 0.214 nM to the mouseprotein.

These results demonstrate that mAb M3-1 (13B1) has a high bindingaffinity for both human and mouse MASP-3.

Demonstration that mAb M3-1 is Capable of Inhibiting Alternative PathwayComplement (APC) Activation and Measurement of the In Vitro Potency ofmAb M3-1

As described in the present disclosure, it has been determined thatMASP-3 is a key regulator of the APC, at least in part due to itsrequirement for the activation of CFD, a central APC enzyme. As alsodescribed in the present disclosure, MASP-3 circulates in the body at arelatively low concentration and has a slow catabolic rate, allowing forlong-lasting inhibition of the pro-inflammatory pathway throughintravenous, subcutaneous and oral routes of MASP-3 antibodyadministration. The following experiment was carried out to determinethe efficacy of mAb M3-1 for inhibiting MASP-3-mediated CFD maturationand inhibition of APC in human serum. Normal human serum containspredominantly active or processed (i.e., mature) CFD, so we performedexperiments in which CFD-depleted human serum (Complement TechnologyA336) was reconstituted with a recombinant, unprocessed form of CFD(pro-CFD). Thus, in this experimental system, APC activation requiresthe processing of pro-CFD into active CFD.

The APC was induced by the addition of zymosan particles, which functionas an activating surface for complement deposition. Varyingconcentrations of mAb M3-1 were added to the serum prior to the additionof recombinant pro-CFD and zymosan. The mixtures were incubated at 37°C. for 75 minutes, and the APC activity was measured by the flowcytometric detection of complement factor Bb (Quidel A252) on thesurface of the zymosan particles.

FIG. 43 graphically illustrates the level of complement factor Bbdeposition on zymosan particles (determined by flow cytometric detectionmeasured in MFI units) in the presence of varying concentrations of mAbM3-1 in CFD-depleted human serum. As shown in FIG. 43, mAb M3-1 showspotent inhibition of the APC in 10% human serum, with an ICso of 0.311nM in this experimental example.

These results demonstrate that MASP-3 plays a key role in APC activationin an in vitro model in human serum, and further demonstrate that mAbM3-1 is a potent inhibitor of the APC.

Inhibition of the APC by mAb M3-1 In Vivo:

In order to determine the efficacy of mAb M3-1 for inhibiting the APC invivo, a group of mice (n =4) received a single intravenous tail veininjection of 10 mg/kg mAb M3-1. Blood collected from the animals wasused to prepare serum, providing a matrix for the flow cytometricassessment of APC activity in an ex vivo assay measuring the level of C3(also C3b and iC3b) deposition on zymosan particles. Serum prepared fromblood harvested at a pre-dose timepoint and multiple post-dose timepoints (96 hrs, 1 week, and 2 weeks) was diluted to 7.5% and zymosanparticles were added to induce the APC. Antibody-treated mice werecompared to a group of control mice (n=4) that were given a singleintravenous dose of vehicle.

FIG. 44 graphically illustrates the level of C3 deposition on zymosanparticles at various time points after a single dose of mAb M3-1 (10mg/kg i.v.) in wild-type mice. As shown in FIG. 44, in the pre-dose timepoint the two conditions show comparable levels of APC activity. At 96hours and the two later time points, the mAb M3-1 treated group showsessentially complete APC inhibition, while the APC activity of thevehicle-treated group remains unabated. As shown in FIG. 44, a singledose of mAb M3-1 administered intravenously to mice led to near-completeablation of systemic APC activity for at least 14 days.

These results demonstrate that mAb M3-1 is a potent inhibitor of the APCin vivo in a mouse model.

Example 12

This Example demonstrates that chimeric mouse V region/human IgG4constant region anti-human MASP-3 monoclonal antibody (mAb M3-1, alsoreferred to as mAb 13B1) provides a clear benefit to survival of redblood cells lacking Crry in a mouse model associated with paroxysmalnocturnal hemoglobinuria (PNH).

Methods:

The chimeric mouse V region/human IgG4 constant region anti-human MASP-3monoclonal antibody (mAb M3-1) was generated as described in Example 11and Example 14. As further described in Example 11, it was determinedthat mAb M3-1 is a potent inhibitor of the APC in a mouse model in vivo.This Example describes the analysis of mAb M3-1 for efficacy in a murinemodel associated with PNH.

Analysis of mAb M3-1 for Efficacy in a Murine Model Associated with PNH

In a mouse model associated with PNH, red blood cells (RBCs) fromCrry-deficient mice lacking the major cell surface repressor of the APCin mouse were obtained for use as donor cells. RBCs obtained from awild-type (WT) donor mouse were run in parallel. These donor RBCs weredifferentially labeled with fluorescent lipophilic dyes (Sigma): WT(red), and Crry-(green). In two different experiments, the labelled WTand Crry- donor cells were mixed 1:1 and injected intravenously intowild-type recipient mice and percent WT and Crry-deficient RBC survival(relative to the early time point) in the recipient mice were determinedby flow cytometric assessment of 20,000 live cell events. In the firstexperiment, multiple pre-dose treatments of mAb M3-1 antibody weregiven, and the effect of the mAb M3-1 was compared to that of anotherinhibitory complement antibody mAb BB5.1 (available from HycultBiotech), which is a C5 inhibitory antibody that has shown efficacy inmultiple mouse studies (Wang et al., PNAS vol 92:8955-8959, 1995; Hugenet al., Kidney Int 71(7):646-54, 2007). Administration of a C5 inhibitoris the current standard of treatment for human patients with PNH. In thesecond experiment, a single pre-treatment dose of mAb M3-1 wasevaluated.

In the first experiment, three different groups of mice (n =4 percondition) were assessed: vehicle-treated condition, mAb M3-1-treatedcondition, and mAb BB5.1 (mAb blocking mouse C5)-treated condition.Labeled cells were injected into mice on “day 0”, and multiple doses ofboth M3-1 and BB5.1 were administered as follows: mAb M3-1 wasadministered intravenously (10 mg/kg) on days −11, −4, −1, and +6. ThemAb BB5.1 was administered by intraperitoneal injection (40 mg/kg) ondays −1, +3, +6, and +10. The vehicle treatment followed the same dosingschedule as mAb M3-1.

FIG. 45 graphically illustrates the percent survival of donor RBCs (WTor Crry-) over a period of 14 days in WT recipient mice treated with mAbM3-1 (10 mg/kg on days −11, 04, −1 and +6), mAb BB5.1 treated, orvehicle treated mice. As shown in FIG. 45, compared to WT RBCs thatshowed survival typical of RBCs in mice in the vehicle-treated animals,Crry-deficient RBCs had rapid clearance (more than 75% cleared within 24hours). Treatment of mice with mAb BB5.1 provided no improvement overvehicle treatment in Crry-deficient RBC survival. In contrast, mAb M3-1treatment caused a dramatic improvement of Crry-deficient RBC survivalover both mAb BB5.1 and vehicle-treated animals. The protective effectof mAb M3-1 was observed throughout the duration of the experiment.

In the second study, differentially labeled WT (red)- and Crry- (green)RBCs were evaluated in two different groups of WT mice (n =4 percondition): vehicle-treated and mAb M3-1-treated. A single dose ofeither vehicle or antibody (20 mg/kg) was given to the recipient mice byintravenous administration six days (day -6) before the labeled donorcells were injected into the recipient mice. The labeled donor RBCs werethen analyzed for percent survival in the recipient mice at incrementaltime points after injection over a 16-day period.

FIG. 46 graphically illustrates the percent survival of donor RBCs (WTor Crry-) over a period of 16 days in WT recipient mice treated with asingle dose of mAb M3-1 (20 mg/kg on day -6) or vehicle-treated mice. Asshown in FIG. 46, a single pre-treatment dose of mAb M3-1 demonstratedimproved survival of Crry-RBCs as compared to the survival of Crry-RBCsin vehicle-treated mice. At 96 hours post injection, approximately 90%of the vehicle-treated WT RBCs survived under the control conditions,whereas only 5% of the Crry-RBCs survived in the vehicle-treated WTmice. In contrast to the vehicle-treated mice, 40% of the Crry-RBCssurvived in the mice treated with mAb M3-1.

Taken together, these results demonstrate that the MASP-3 inhibitoryantibody mAb M3-1 provides a clear benefit to survival of RBCs lackingCrry, a key surface complement inhibitor in a mouse model associatedwith PNH.

Example 13

This Example describes a study demonstrating that a chimeric MASP-3inhibitory monoclonal antibody (mAb M3-1, also referred to as mAb 13B1)reduces clinical scores in collagen antibody-induced arthritis (CAIA), amurine model of rheumatoid arthritis (RA).

Background/Rationale:

CAIA is a well-established animal model of arthritis. In additional toproviding insight into RA, the pathology of the CAIA model has anestablished connection with the APC. Banda and coworkers havedemonstrated improved outcomes in the CAIA model in mice carryingdeficiencies in components of the APC, such as factor B and factor D(Banda et al., J. Immunol vol 177:1904-1912, 2006 and Banda et al.,Clinical & Exp Imunol vol 159:100-108, 2009). APC mouse knock-outsdemonstrate lower arthritis (disease) scores, lower incidence, and lessC3 and factor H deposition in synovium and surrounding tissues relativeto WT controls. Additionally, disease activity scores, complement C3tissue deposition in the joint, and histopathologic injury scores weremarkedly decreased in MASP1/3 knock-out mice (Banda et al., J Immunolvol 185:5598-5606, 2010). Therefore, the MASP-3 inhibitory antibody mAbM3-1 was analyzed for efficacy in the CAIA.

Methods:

The chimeric MASP-3 monoclonal antibody (mAb M3-1) was generated asdescribed in Example 11 and Example 14. As further described in Example11, it was determined that mAb M3-1 is a potent inhibitor of the APC ina mouse model in vivo.

mAb M3-1 was tested in the CAIA model as follows. Wild-type mice (n=7)were injected intravenously with 3 mg of a cocktail of anti-collagenantibodies on day 0. The mice were dosed intraperitoneally with E. colilipopolysaccharide (LPS) (25 μg/mouse) on day +3. As described inNandakumar et al. (Am J Pathol 163(5):1827-1837, 2003), arthritistypically occurs in this model on days +3 through +10. Terminal serumsamples were collected on day +14. mAb M3-1 (5 mg/kg and 20 mg/kg) wasdosed on days −12, −5, +1 and day +7. Vehicle (PBS) was injected as anegative control.

Clinical scores were evaluated for each mouse on all 4 paws on studydays 0 through 14 using the following scoring standards:

-   0=normal-   1=1 hind and/or fore paw joint affected or minimal diffuse erythema    and swelling-   2=2 hind and/or fore paw joints affected or mild diffuse erythema    and swelling-   3=3 hind and/or fore paw joints affected or moderate diffuse    erythema and swelling-   4=marked diffuse erythema and swelling, or 4 digit joints affected-   5=severe diffuse erythema and severe swelling of entire paw, unable    to flex digits.

The incidence=% mice within a treatment group showing arthritic symptomswas also determined.

The results are shown in FIG. 47 (clinical scores) and FIG. 48(incidence of arthritis). FIG. 47 graphically illustrates the clinicalscores of the mice treated with mAb M3-1 (5 mg/kg or 20 mg/kg) orvehicle over a 14-day time course. FIG. 48 graphically illustrates thepercent incidence of arthritis of the mice treated with mAb M3-1 (5mg/kg or 20 mg/kg) or vehicle over a 14-day time course. As shown inFIG. 47, mAb M3-1 demonstrates a clear therapeutic benefit for bothendpoints starting at day 5 and lasting throughout the duration of thestudy. As shown in FIG. 48, while the incidence of disease reached 100%in the vehicle-treated animals, two-thirds of the animals in the 5 mg/kgmAb M3-1 condition remained disease-free. Additionally, only one of theanimals (i.e., only one in a total n=7) demonstrated any arthriticsymptoms in the 20 mg/kg mAb M3-1 condition.

The results of this study demonstrate that the MASP-3 inhibitoryantibody mAb M3-1 provides a clear therapeutic benefit in the CAIAmodel, a well-established murine model of rheumatoid arthritis (RA) anda model strongly linked to APC activation. As shown in Example 11, asingle dose of mAb M3-1 administered intravenously to mice led tonear-complete ablation of systemic APC activity for at least 14 days. Asshown in this Example, in the animal model induced by administration ofauto-antibodies against mouse connective tissue, mAb M3-1 reduced theincidence and severity of clinical arthritis scores in a dose-dependentfashion. Compared to control-treated animals, mAb M3-1 reduced theincidence and severity of the disease by approximately 80% at thehighest dose tested. Therefore, it is expected that administration of aMASP-3 inhibitory antibody, such as mAb M3-1 will be an effectivetherapy in patients suffering from arthritis, such as rheumatoidarthritis, osteoarthritis, juvenile rheumatoid arthritis,infection-related arthritis, psoriatic arthritis, as well as ankylosingspondylitis and Bechcet's disease.

Example 14

This Example describes the generation of high affinity anti-human MASP-3inhibitory antibodies.

Background/Rationale:

A limited number of antibodies specific for MASP-3 have been described(Thiel et al., Mol. Immunol. 43:122, 2006; Moller-Kristensen et al.,Int. Immunol. 19:141, 2006; Skjoedt et al., Immunobiol 215:921, 2010).These antibodies were useful for detection assays such as Westernblotting, immunoprecipitation, and as capture or detection reagents inELISA assays. However, the antibodies described in Thiel et al., 2006,Moller-Kristensen et al., 2006 and Skjoedt et al., 2010 have not beenfound to inhibit MASP-3 catalytic activity.

MASP-3 antibodies were also generated previously, as described inExample 7 herein (also published as Example 15 in WO2013/192240) byscreening a chicken antibody library in a modified DT40 cell line,DTLacO, for MASP-3 binding molecules. These antibodies bound to humanMASP-3 in the nanomolar range with an EC₅₀ between 10 nM and 100 nM andpartially inhibited cleavage of pro-CFD by MASP-3.

This Example describes the generation of anti-human MASP-3 inhibitoryantibodies with unusually strong binding affinity (i.e., subnanomolarbinding affinity, ranging from ≤500 pM to 20 pM). The antibodiesdescribed in this Example specifically bind to human MASP-3 with highaffinity (e.g., ≤500 pM), inhibit Factor D maturation, and do not bindto human MASP-1 (SEQ ID NO:8).

Methods: 1. Generation of Chimeric Mouse V Region/Human IgG ConstantRegion Anti-Human MASP-3 Monoclonal Antibodies

Seven to fourteen-week old C57BL/6, MASP-1/3 knockout mice wereimmunized with either the human MASP-3 CCP1/CCP2/SP polypeptide (aminoacid residues 299-728 of SEQ ID NO:2) including a StrepTag II epitopetag on the N-terminus; or were immunized with the human MASP-3 SP domain(amino acid residues 450-728 of SEQ ID NO:2), including StrepTagll onthe N-terminus, using the Sigma Adjuvant System (Sigma-Aldrich, StLouis, Mo.). The mice were injected intraperitoneally with 50 μg ofimmunogen per mouse. The immunized mice were boosted 14 days later withadditional immunogen in adjuvant. Thereafter, for several weeks, themice were boosted every 14 to 21 days with immunogen in PBS. Serumsamples from the mice were periodically prepared from tail bleeds andtested by ELISA for the presence of antigen-specific antibodies. Micewith a significant antibody titer received a pre-fusion immunogen boostin PBS four days prior to splenic fusion. Three days prior to thefusion, the mice were treated subcutaneously at the base of the tailwith 50 μg of a anti-CD40 agonist mAb in PBS (R&D Systems, Minneapolis,MN) to increase B cells numbers (see Rycyzyn et al., Hybridoma 27:25-30,2008). The mice were sacrificed and the spleen cells were harvested andfused to a selected murine myeloma cell line P3/NSI/1-AG4-1 (NS-1) (ATCCNo. TIB18) using 50% polyethylene glycol or 50% polyethylene glycol plus10% DMSO. The fusions generated hybridoma cells which were plated in 96well tissue culture plates containing HAT (hypoxanthine, aminopterin andthymidine) medium to inhibit proliferation of non-fused cells, myelomahybrids and spleen hybrids. After hybridoma selection, the culturesupernatants were assayed for MASP-3 binding (ELISA) and inhibition ofpro-Factor D activation. The positive hybridomas were identified andsubcloned by serial dilution methods.

TABLE 17 Summary of Fusion Experiments MASP-3 MASP-3 Immunogen: TotalBinding Functional Fusion Human MASP-3 hybridomas hybridomas hybridomas1 SP 434 38 10 2 SP 279 13 0 3 CCP1/CCP2/SP 348 40 2 4 CCP1/CCP2/SP 31960 2 5 CCP1/CCP2/SP 651 152 1 6 CCP1/CCP2/SP 1297 ND 1 Note: “ND” meansthis fusion was only screened for functional inhibition of pro-CFDactivation.

Results:

As shown in TABLE 17, a total of 3328 hybridomas from immunized MASP1/3KO mice were screened, of which >303 were found to bind to MASP-3 and ofwhich 16 were found to bind to MASP-3 and to inhibit pro-CFD activation.mAb M3-1 (13B1) described in Example 11 is one of the 16 functionalMASP-3 inhibitory antibodies described in TABLE 17. As described inExample 15, it was determined that all 16 functional MASP-3 inhibitoryantibodies bind to human MASP-3 with unusually strong binding affinity(≤500 pM).

Discussion:

This Example describes the generation of antibodies that inhibit humanMASP-3 with unusually strong binding affinity (i.e., subnanomolarbinding affinity, ranging from ≤500 pM to 20 pM) by immunizing MASP1/3knockout mice. The antibodies described in this Example specificallybind to human MASP-3 with high affinity (e.g., ≤500 pM), inhibit FactorD maturation, and do not bind to human MASP-1. As described herein, theamino acid sequences of human, mouse and chicken MASP-3 revealed thatthe SP domain of MASP-3 is highly conserved, especially in the activesite (see FIGS. 4 and 5). It is likely that the ability to generateMASP-3 inhibitory antibodies with unusually strong binding affinity inMASP1/3 KO mice, as described in this example, is due in part toavoidance of immunological tolerance that may hamper the generation ofhighly potent MASP-3 catalytic site-specific antibodies in wild-typeanimals.

Example 15

This Example describes the cloning and sequence analysis of highaffinity anti-human MASP-3 inhibitory mAbs.

Methods:

Cloning and Purification of Recombinant Antibodies:

The heavy chain and light chain variable regions were cloned from thehybridomas described in Examples 11 and 14 using RT-PCR and weresequenced. Mouse-human chimeric mAbs consisting of the mouse mAbvariable regions fused to the human IgG4 heavy chain (SEQ ID NO:311) andkappa light chain (SEQ ID NO:313) constant regions were produced asrecombinant proteins in Expi293F cells. The IgG4 constant hinge region(SEQ ID NO:311) contains the stabilizing S228P amino acid substitution.In one embodiment, the chimeric mAbs were fused to the human IgG4constant hinge region (SEQ ID NO:312) which contains the S228P aminoacid substitution and also a mutation that promotes FcRn interations atlow pH.

The sequences of the heavy chain variable regions and light chainvariable regions are shown in FIGS. 49A and 49B, respectively(“SIN”=“SEQ ID NO:” in FIG. 49A and FIG. 49B), and are included below.The complementarity regions (CDRs) and framework regions (FRs) of eachare provided in TABLES 18-22 below.

FIG. 50A is a dendrogram of the VH regions of high affinity anti-humanMASP-3 inhibitory mAbs generated in MASP1/3 KO mice. FIG. 50B is adendrogram of the VL regions of high affinity anti-human MASP-3inhibitory mAbs generated in MASP1/3 KO mice. As shown in FIGS. 50A and50B, several groups of related antibodies were identified.

Presented below is the heavy chain variable region (VH) sequence foreach high affinity MASP-3 inhibitory antibody. The Kabat CDRs areunderlined.

Heavy Chain Variable Regions:

4D5_VH:  SEQ ID NO: 24 QVQLKQSGPELVKPGASVKLSCKASGYTFTTDDINWVKQRPGQGLEWIGWIYPRDDRTKYNDKFKDKATLTVDTSSNTAYMDLHSLTSEDSAVYFC SSLEDTYWGQGTLVAVSS1F3_VH:  SEQ ID NO: 25 QVQLKQSGPELVKPGASVKLSCKASGYTFTSNDINWVKQRPGQGLEWIGWIYPRDGSIKYNEKFTDKATLTVDVSSSTAYMELHSLTSEDSAVYFC SGVEDSYWGQGTLVTVSS4B6_VH:  SEQ ID NO: 26 QVQLKQSGPELVKPGASVKLSCKASGYTFTSNDINWVKQRPGQGLEWIGWIYPRDGTTKYNEEFTDKATLTVDVSSSTAFMELHSLTSEDSAVYFC SSVEDSYWGQGTLVTVSS1A10_VH:  SEQ ID NO: 27 QVQLKQSGPELVKPGASVKLSCKASGYTFTSNDINWVKQRPGQGLEWIGWIYPRDGTTKYNEKFTDKATLTVDVSSSTAFMELHRLTSEDSAVYFC SSVEDSYWGQGTLVTVSS10D12_VH:  SEQ ID NO: 28QIQLVQSGPELKKPGETVKISCKASGYIFTSYGMSWVRQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSARTPYLQINNLKNEDTATYFC ARGGEAMDYWGQGTSVTVSS35C1_VH:  SEQ ID NO: 29 QIQLVQSGPELKTPGETVKISCKASGYIFTSYGITWVKQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTTTYFC TRGGDALDYWGQGTSVTVSS13B1_VH:  SEQ ID NO: 30 QVQLKQSGAELMKPGASVKLSCKATGYTFTGKWIEWVKQRPGHGLEWIGEILPGTGSTNYNEKFKGKATFTADSSSNTAYMQLSSLTTEDSAMYYC LRSEDVWGTGTTVTVSS1G4_VH:  SEQ ID NO: 31 QVQLKQSGAELMKPGASVKLACKATGYTFTGYWIEWIKQRPGQGLEWIGEMLPGSGSTHYNEKFKGKATFTADTSSNTAYMQLSGLTTEDSAIYYC VRSIDYWGQGTTLTVSS1E7_VH:  SEQ ID NO: 32 QVQLKQSGPELARPWASVKISCQAFYTFSRRVHFAIRDTNYWMQWVKQRPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCASGSHYFDYWGQGTTLTVSS 2D7_VH:  SEQ ID NO: 33EVQLQQSGPELVKPGASVKVSCKASGYTLTDYYMNWVKQSHGKSLEWIGDVNPNNDGTTYNQKFKGRATLTVDKSSNTASMELRSLTSEDSAVYYCAICPFYYLGKGTHFDYWGQGTSLTVSS 49C11_VH: SEQ ID NO: 34EVQLQQSGPVLVKPGASGKMSCKASGYKFTDYYMIWVKQSHGKSLEWIGVIKIYNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARGPSLYDYDPYWYFDVWGTGTTVTVSS 15D9_VH: SEQ ID NO: 35QVQLKQSGTELMKPGASVNLSCKASGYTFTAYWIEWVKQRPGHGLEWIGEILPGSGTTNYNENFKDRATFTADTSSNTAYMQLSSLTSEDSAIYYCARSYYYASRWFAFWGQGTLVTVSS 2F5_VH:  SEQ ID NO: 36EVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVKQRPGQGLEWIGDIYPGSGSTNYNEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCARRRYYATAWFAYWGQGTLVTVSS 1B11_VH: SEQ ID NO: 37QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPGSGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARNYYISSPWFAYWGQGTLVTVSS 2F2_VH: SEQ ID NO: 38QVQLKQSGAELVTPGASVKMSCKASGYTFTTYPIEWMKQNHGKSLEWIGNFHPYNDDTKYNEKFKGKATLTVEKSSNTVYLELSRLTSDDSAVYFCARRVYYSYFWFGYWGHGTLVTVSS 11B6_VH:  SEQ ID NO: 39QVQLKQSGAELVKPGASVKMSCKASGYTFTTYPIEWMKQNHGKSLEWIGNFHPYNGDSKYNEKFKGKATLTVEKSSSTVYLELSRLPSADSAIYYCARRHYAASPWFAHWGQGTLVTVSS

TABLE 18 MASP-3 Antibody VH Sequences  (CDRs and FR regions, Kabat)Anti- body HC FR1 HC CDR1 4D5 QVQLKQSGPELVKPGASVKLSCK TDDINASGYTFT(SEQ ID NO: 55) (SEQ ID NO: 56) 1F3 QVQLKQSGPELVKPGASVKLSCK SNDINASGYTFT(SEQ ID NO: 55) (SEQ ID NO: 62) 4B6 QVQLKQSGPELVKPGASVKLSCK SNDINASGYTFT(SEQ ID NO: 55) (SEQ ID NO: 62) 1A10 QVQLKQSGPELVKPGASVKLSCKSNDIN ASGYTFT(SEQ ID NO: 55) (SEQ ID NO: 62) 10D12QIQLVQSGPELKKPGETVKISCK SYGMS ASGYIFT(SEQ ID NO: 71) (SEQ ID NO: 72)35C1 QIQLVQSGPELKTPGETVKISCK SYGIT ASGYIFT(SEQ ID NO: 78)(SEQ ID NO: 79) 13B1 QVQLKQSGAELMKPGASVKLSCK GKWIEATGYTFT(SEQ ID NO: 83) (SEQ ID NO: 84) 1G4 QVQLKQSGAELMKPGASVKLACK GYWIEATGYTFT(SEQ ID NO: 90) (SEQ ID NO: 91) 2F5 EVQLQQPGAELVKPGASVKMSCK SYWITASGYTFT(SEQ ID NO: 97) (SEQ ID NO: 98) 1B11 QVQLKQSGAELVRPGASVKLSCKDYYIN ASGYTFT(SEQ ID NO: 102) (SEQ ID NO: 103) 1E7QVQLKQSGPELARPWASVKISCQ RVHFAIRDTNYWMQ AFYTFSR(SEQ ID NO: 108)(SEQ ID NO: 109) 2F2 QVQLKQSGAELVTPGASVKMSCK TYPIEASGYTFT(SEQ ID NO: 113) (SEQ ID NO: 114) 11B6 QVQLKQSGAELVKPGASVKMSCKTYPIE ASGYTFT(SEQ ID NO: 120) (SEQ ID NO: 114) 2D7EVQLQQSGPELVKPGASVKVSCK DYYMN ASGYTLT(SEQ ID NO: 124) (SEQ ID NO: 125)49C11 EVQLQQSGPVLVKPGASGKMSCK DYYMI ASGYKFT(SEQ ID NO: 131)(SEQ ID NO: 132) 15D9 QVQLKQSGIELMKPGASVNLSCK AYWIEASGYTFT(SEQ ID NO: 136) (SEQ ID NO: 137) Anti- body HC FR2 HC CDR2 4D5WVKQRPGQGLEWIG WIYPRDDRTKYNDKFKD (SEQ ID NO: 57) (SEQ ID NO: 58) 1F3WVKQRPGQGLEWIG WIYPRDGSIKYNEKFTD (SEQ ID NO: 57) (SEQ ID NO: 63) 4B6WVKQRPGQGLEWIG WIYPRDGTTKYNEEFTD (SEQ ID NO: 57) (SEQ ID NO: 67) 1A10WVKQRPGQGLEWIG WIYPRDGTTKYNEKFTD (SEQ ID NO: 57) (SEQ ID NO: 69) 10D12WVRQAPGKGLKWMG WINTYSGVPTYADDFKG (SEQ ID NO: 73) (SEQ ID NO: 74) 35C1WVKQAPGKGLKWMG WINTYSGVPTYADDFKG (SEQ ID NO: 80) (SEQ ID NO: 74) 13B1WVKQRPGHGLEWIG EILPGTGSTNYNEKFKG (SEQ ID NO: 85) (SEQ ID NO: 86) 1G4WIKQRPGQGLEWIG EMLPGSGSTHYNEKFKG (SEQ ID NO: 92) (SEQ ID NO: 93) 2F5WVKQRPGQGLEWIG DIYPGSGSTNYNEKFKS (SEQ ID NO: 57) (SEQ ID NO: 99) 1B11WVKQRPGQGLEWIA RIYPGSGNTYYNEKFKG (SEQ ID NO: 104) (SEQ ID NO: 105) 1E7WVKQRPGQGLEWIG AIYPGNGDTSYNQKFKG (SEQ ID NO: 57) (SEQ ID NO: 110) 2F2WMKQNHGKSLEWIG NFHPYNDDTKYNEKFKG (SEQ ID NO: 115) (SEQ ID NO: 116) 11B6WMKQNHGKSLEWIG NFHPYNGDSKYNEKFKG (SEQ ID NO: 115) (SEQ ID NO: 121) 2D7WVKQSHGKSLEWIG DVNPNNDGTTYNQKFKG (SEQ ID NO: 126) (SEQ ID NO: 127) 49C11WVKQSHGKSLEWIG VIKIYNGGTSYNQKFKG (SEQ ID NO: 126) (SEQ ID NO: 133) 15D9WVKQRPGHGLEWIG EILPGSGTTNYNENFKD (SEQ ID NO: 85) (SEQ ID NO: 138) Anti-body HC FR3 HC CDR3 4D5 KATLTVDTSSNTAYMDLHSLTSE LEDTYDSAVYFCSS(SEQ ID NO: 59) (SEQ ID NO: 60) 1F3 KATLTVDVSSSTAYMELHSLTSEVEDSY DSAVYFCSG(SEQ ID NO: 64) (SEQ ID NO: 65) 4B6KATLTVDVSSSTAFMELHSLTSE VEDSY DSAVYFCSS(SEQ ID NO: 68) (SEQ ID NO: 65)1A10 KATLTVDVSSSTAFMELHRLTSE VEDSY DSAVYFCSS(SEQ ID NO: 70)(SEQ ID NO: 65) 10D12 RFAFSLETSARTPYLQINNLKNE GGEAMDYDTATYFCAR(SEQ ID NO: 75) (SEQ ID NO: 76) 35C1 RFAFSLETSASTAYLQINNLKNEGGDALDY DTTTYFCTR(SEQ ID NO: 81) (SEQ ID NO: 82) 13B1KATFTADSSSNTAYMQLSSLTTE SEDV DSAMYYCLR(SEQ ID NO: 87) (SEQ ID NO: 88)1G4 KATFTADTSSNTAYMQLSGLTTE SIDY DSAIYYCVR(SEQ ID NO: 94)(SEQ ID NO: 95) 2F5 KATLTVDTSSSTAYMQLSSLTSE RRYYATAWFAYDSAVYYCAR(SEQ ID NO: 100) (SEQ ID NO: 101) 1B11 KATLTAEKSSSTAYMQLSSLTSENYYISSPWFAY DSAVYFCAR(SEQ ID NO: 106) (SEQ ID NO: 107) 1E7KATLTADKSSSTAYMQLSSLTSE GSHYFDY DSAVYYCAS(SEQ ID NO: 111)(SEQ ID NO: 112) 2F2 KATLTVEKSSNTVYLELSRLTSD RVYYSYFWFGYDSAVYFCAR(SEQ ID NO: 117) (SEQ ID NO: 118) 11B6 KATLTVEKSSSTVYLELSRLPSARHYAASPWFAH DSAIYYCAR(SEQ ID NO: 122) (SEQ ID NO: 123) 2D7RATLTVDKSSNTASMELRSLTSE CPFYYLGKGTHFDY DSAVYYCAI(SEQ ID NO: 128)(SEQ ID NO: 129) 49C11 KATLTVDKSSSTAYMELNSLTSE GPSLYDYDPYWYFDVDSAVYYCAR(SEQ ID NO: 134) (SEQ ID NO: 135) 15D9 RATFTADTSSNTAYMQLSSLTSESYYYASRWFAF DSAIYYCAR(SEQ ID NO: 139) (SEQ ID NO: 140) Anti- body HC FR44D5 WGQGTLVAVSS (SEQ ID NO: 61) 1F3 WGQGTLVTVSS (SEQ ID NO: 66) 4B6WGQGTLVTVSS (SEQ ID NO: 66) 1A10 WGQGTLVTVSS (SEQ ID NO: 66) 10D12WGQGTSVTVSS (SEQ ID NO: 77) 35C1 WGQGTSVTVSS (SEQ ID NO: 77) 13B1WGTGTTVTVSS (SEQ ID NO: 89) 1G4 WGQGTTLTVSS (SEQ ID NO: 96) 2F5WGQGTLVTVSS (SEQ ID NO: 66) 1B11 WGQGTLVTVSS (SEQ ID NO: 66) 1E7WGQGTTLTVSS (SEQ ID NO: 96) 2F2 WGHGTLVTVSS (SEQ ID NO: 119) 11B6WGQGTLVTVSS (SEQ ID NO: 66) 2D7 WGQGTSLTVSS (SEQ ID NO: 130) 49C11WGTGTTVTVSS (SEQ ID NO: 89) 15D9 WGQGTLVTVSS (SEQ ID NO: 66)

Presented below are the light chain variable region (VL) sequences forthe high affinity MASP-3 inhibitory antibodies. The Kabat CDRs areunderlined. These regions are the same whether numbered by the Kabat orChothia system.

Light Chain Variable Regions:

4D5_VL:  SEQ ID NO: 40DIVMTQSPSSLAVSAGEKVTMTCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFSLTISSVQAEDLAVYYCKQSYNLYT FGGGTKLEIKR 1F3_VL: SEQ ID NO: 41 DIVMTQSPSSLAVSAGERVTMSCKSSQSLLISRTRKNYLSWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYT FGGGTKLEIKR4B6_VL: (SAME for 1A10 VL) SEQ ID NO: 42DIVMTQTSPSSLAVSAGEKVTMSCKSSQSLLISRTRKNYLSWYQQKPGQSPKLLITYWASRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLY TFGGGTKLEIKR10D12_VL:  SEQ ID NO: 43DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWT FGGGTKLEIKR35C1_VL:  SEQ ID NO: 44DIVMTQAPLTLSVTIGQPASISCKSSQSLLDSDGKTYLSWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYT FGGGTKLEIKR13B1_VL:  SEQ ID NO: 45DIVMTQSPSSLAVSAGEKVTMSCKSSOSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNIPT FGGGTKLEIKR 1G4_VL: SEQ ID NO: 46 DVLMTQTPLSLPVSLGEQASISCRSSQSLVQSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPT FGGGTKLEIKR 1E7_VL: SEQ ID NO: 47 DIQLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGGGT KLEIKR 2D7_VL: SEQ ID NO: 48 DIQMTQTPASLSASLGDRVTISCRASCIDISNFLNWYQQKPNGTVKLLVFYTSRLHSGVPSRFSGSGSGAEHSLTISNLEQEDVATYFCQQGFTLPWTFGGG TKVEIKR 49C11_VL:SEQ ID NO: 49 DVLMTQTPLSLPVSLGDQASFSCRSSQSLIHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWT FGGGTKLEIKR15D9_VL:  SEQ ID NO: 50DIVMTQSQKFMSTSIGDRVSVTCRASONVGPNLAWYQQKPGQSPKALIYSASYRFSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNRYPFTFGSGT KLEIKR 2F5_VL: SEQ ID NO: 51 DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLISSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDVADYFCQQYNSYPLTFGAGT KLELKR 1B11_VL: SEQ ID NO: 52 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYNRYPLTFGAGT KLELKR 2F2_VL: SEQ ID NO: 53 DIVMTQSQKFMSTSVGDRVNVTCKASQNVGTHVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPRALTFGA GTKLELKR 11B6_VL: SEQ ID NO: 54 DIVMTQSQKFMSTSVGDRVNVTCKASQNVGTHVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVHSEDLAEYFCQQYNSYPFTFGSGT KLEIKR

TABLE 19 MASP-3 Antibody VL Sequences(CDRs and FR regions, Kabat and Chothia) Anti- body LC FR1 LC CDR1 4D5DIVMTQSPSSLAVSAGEKVTMTC KSSQSLLNSRTRKNYLA (SEQ ID NO: 141)(SEQ ID NO: 142) 1F3 DIVMTQSPSSLAVSAGERVTMSC KSSQSLLISRTRKNYLS(SEQ ID NO: 148) (SEQ ID NO: 149) 4B6 DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLISRTRKNYLS (SEQ ID NO: 151) (SEQ ID NO: 149) 1A10* [used 4B6 LC:[used 4B6 LC: SEQ ID NO: 151] SEQ ID NO: 149] 10D12DVLMTQTPLTLSVTIGQPASISC KSSQSLLDSDGKTYLN (SEQ ID NO: 152)(SEQ ID NO: 153) 35C1 DIVMTQAPLTLSVTIGQPASISC KSSQSLLDSDGKTYLS(SEQ ID NO: 158) (SEQ ID NO: 159) 13B1 DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLA (SEQ ID NO: 151) (SEQ ID NO: 142) 1G4DVLMTQTPLSLPVSLGEQASISC RSSQSLVQSNGNTYLH (SEQ ID NO: 162)(SEQ ID NO: 163) 2F5 DIVMTQSQKFMSTSVGDRVSITC KASQNVGTAVA(SEQ ID NO: 168) (SEQ ID NO: 169) 1B11 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVA (SEQ ID NO: 175) (SEQ ID NO: 176) 1E7DIQLTQSPAILSVSPGERVSFSC RASQSIGTSIH (SEQ ID NO: 181) (SEQ ID NO: 182)2F2 DIVMTQSQKFMSTSVGDRVNVTC KASQNVGTHVA (SEQ ID NO: 187)(SEQ ID NO: 188) 11B6 DIVMTQSQKFMSTSVGDRVNVTC KASQNVGPTVA(SEQ ID NO: 187) (SEQ ID NO: 191) 2D7 DIQMTQTPASLSASLGDRVTISCRASQDISNFLN (SEQ ID NO: 195) (SEQ ID NO: 196) 49C11DVLMTQTPLSLPVSLGDQASFSC RSSQSLIHSNGNTYLH (SEQ ID NO: 202)(SEQ ID NO: 203) 15D9 DIVMTQSQKFMSTSIGDRVSVTC RASQNVGPNLA(SEQ ID NO: 205) (SEQ ID NO: 206) Anti- body LC FR2 LC CDR2 4D5WYQQKPGQSPKLLIY WASTRES (SEQ ID NO: 143) (SEQ ID NO: 144) 1F3WYQQKPGQSPKLLIY WASTRES (SEQ ID NO: 143) (SEQ ID NO: 144) 4B6WYQQKPGQSPKLLIY WASTRES (SEQ ID NO: 143) (SEQ ID NO: 144) 1A10[used 4B6 LC: [used 4B6 LC: SEQ ID NO: 143] SEQ ID NO: 144] 10D12WLLQRPGQSPKRLIY LVSKLDS (SEQ ID NO: 154) (SEQ ID NO: 155) 35C1WLLQRPGQSPKRLIY LVSKLDS (SEQ ID NO: 154) (SEQ ID NO: 155) 13B1WYQQKPGQSPKLLIY WASTRES (SEQ ID NO: 143) (SEQ ID NO: 144) 1G4WYLQKPGQSPKLLIY KVSNRFS (SEQ ID NO: 164) (SEQ ID NO: 165) 2F5WYQQKPGQSPKLLIS SASNRYT (SEQ ID NO: 170) (SEQ ID NO: 171) 1B11WYQQKPGQSPKALIY SASYRYS (SEQ ID NO: 177) (SEQ ID NO: 178) 1E7WYQQRTNGSPRLLIK YASESIS (SEQ ID NO: 183) (SEQ ID NO: 184) 2F2WYQQKPGQSPKALIY SASYRYS (SEQ ID NO: 177) (SEQ ID NO: 178) 11B6WYQQKPGQSPKALIY SASYRYS (SEQ ID NO: 177) (SEQ ID NO: 178) 2D7WYQQKPNGTVKLLVF YTSRLHS (SEQ ID NO: 197) (SEQ ID NO: 198) 49C11WYLQKPGQSPKLLIY KVSNRFS (SEQ ID NO: 164) (SEQ ID NO: 165) 15D9WYQQKPGQSPKALIY SASYRFS (SEQ ID NO: 177) (SEQ ID NO: 207) Anti- bodyLC FR3 LC CDR3 4D5 GVPDRFTGSGSGTDFSLTISSVQAE KQSYNLYTDLAVYYC(SEQ ID NO: 145) (SEQ ID NO: 146) 1F3 GVPDRFTGSGSGTDFTLTISSVQAEKQSYNLYT DLAVYYC(SEQ ID NO: 150) (SEQ ID NO: 146) 4B6GVPDRFTGSGSGTDFTLTISSVQAE KQSYNLYT DLAVYYC(SEQ ID NO: 150)(SEQ ID NO: 146) 1A10 [used 4B6 LC: [used 4B6 LC: SEQ ID NO: 150]SEQ ID NO: 146] 10D12 GVPDRFTGSGSGTDFTLKISRVEAE WQGTHFPWTDLGVYYC(SEQ ID NO: 156) (SEQ ID NO: 157) 35C1 GVPDRFTGSGSGTDFTLKISRVEAEWQGTHFPYT DLGVYYC(SEQ ID NO: 156) (SEQ ID NO: 160) 13B1GVPDRFTGSGSGTDFTLTISSVQAE KQSYNIPT DLAVYYC(SEQ ID NO: 150)(SEQ ID NO: 161) 1G4 GVPDRFSGSGSGTDFTLKISRVEAE SQSTHVPPTDLGVYFC(SEQ ID NO: 166) (SEQ ID NO: 167) 2F5 GVPDRFTGSGSGTDFTLTISNMQSEQQYNSYPLT DVADYFC(SEQ ID NO: 172) (SEQ ID NO: 173) 1B11GVPDRFTGSGSGTDFTLTISNVQSE QQYNRYPLT DLADYFC(SEQ ID NO: 179)(SEQ ID NO: 180) 1E7 GIPSRFSGSGSGTDFTLSINSVESE QQSNSWPYTDIADYYC(SEQ ID NO: 185) (SEQ ID NO: 186) 2F2 GVPDRFTGSGSGTDFTLTISNVQSEQQYNSYPRALT DLAEYFC(SEQ ID NO: 189) (SEQ ID NO: 190) 11B6GVPDRFTGSGSGTDFTLTISNVHSE QQYNSYPFT DLAEYFC(SEQ ID NO: 192)(SEQ ID NO: 193) 2D7 GVPSRFSGSGSGAEHSLTISNLEQE QQGFTLPWTDVATYFC(SEQ ID NO: 199) (SEQ ID NO: 200) 49C11 GVPDRFSGSGSGTDFTLKISRVEAESQSTHVPWT DLGVYFC(SEQ ID NO: 166) (SEQ ID NO: 204) 15D9GVPDRFTGSGSGTDFTLTISNVQSE QQYNRYPFT DLAEYFC(SEQ ID NO: 189)(SEQ ID NO: 208) Anti- body LC FR4 4D5 FGGGTKLEIKR (SEQ ID NO: 147) 1F3FGGGTKLEIKR (SEQ ID NO: 147) 4B6 FGGGTKLEIKR (SEQ ID NO: 147) 1A10[used 4B6 LC: SEQ ID NO: 147] 10D12 FGGGTKLEIKR (SEQ ID NO: 147) 35C1FGGGTKLEIKR (SEQ ID NO: 147) 13B1 FGGGTKLEIKR (SEQ ID NO: 147) 1G4FGGGTKLEIKR (SEQ ID NO: 147) 2F5 FGAGTKLELKR (SEQ ID NO: 174) 1B11FGAGTKLELKR (SEQ ID NO: 174) 1E7 FGGGTKLEIKR (SEQ ID NO: 147) 2F2FGAGTKLELKR (SEQ ID NO: 174) 11B6 FGSGTKLEIKR (SEQ ID NO: 194) 2D7FGGGTKVEIKR (SEQ ID NO: 201) 49C11 FGGGTKLEIKR (SEQ ID NO: 147) 15D9FGSGTKLEIKR (SEQ ID NO: 194) *Note: the light chain for mAb 1A10 was notidentified, so the light chain from 4B6 was used with the 1A10 HC.

TABLE 20 Consensus Sequences for Group IA HC CDRs: Antibody RegionSequence 4D5 HC-CDR1 TDDIN (SEQ ID NO: 56) 1F3 HC-CDR1 SNDIN(SEQ ID NO: 62) 4B6 HC-CDR1 SNDIN (SEQ ID NO: 62) 1A10 HC-CDR1 SNDIN(SEQ ID NO: 62) Consensus HC-CDR1 XXDIN (SEQ ID NO: 209) whereinX at position 1 is S or T; and X at position 2 is N or D 4D5 HC-CDR2WIYPRDDRTKYNDKFKD (SEQ ID NO: 58) 1F3 HC-CDR2 WIYPRDGSIKYNEKFTD(SEQ ID NO: 63) 4B6 HC-CDR2 WIYPRDGTTKYNEEFTD (SEQ ID NO: 67) 1A10HC-CDR2 WIYPRDGTTKYNEKFTD (SEQ ID NO: 69) Consensus HC-CDR2WIYPRDXXXKYNXXFXD (SEQ ID NO: 210) wherein X at position 7 is G or D;X at position 8 is S, T or R; X at position 9 is I or T;X at position 13 is E or D; X at position 14 is K or E;X at position 16 is T or K 4D5 HC-CDR3 LEDTY (SEQ ID NO: 60) 1F3 HC-CDR3VEDSY (SEQ ID NO: 65) 4B6 HC-CDR3 VEDSY (SEQ ID NO: 65) 1A10 HC-CDR3VEDSY (SEQ ID NO: 65) Consensus HC-CDR3 XEDXY (SEQ ID NO: 211)wherein X at position 1 is L or V, and wherein X at position 4 is T or S

TABLE 21 Consensus Sequences for Group IA LC CDRs: Antibody RegionSequence 4D5 LC-CDR1 KSSQSLLNSRTRKNYLA (SEQ ID NO: 142) 4D5-NQ LC-CDR1KSSQSLLQSRTRKNYLA (SEQ ID NO: 257) 4D5-NA LC-CDR1 KSSQSLLASRTRKNYLA(SEQ ID NO: 258) 4D5-ST LC-CDR1 KSSQSLLNTRTRKNYLA (SEQ ID NO: 259) 1F3LC-CDR1 KSSQSLLISRTRKNYLS (SEQ ID NO: 149) 4B6 LC-CDR1 KSSQSLLISRTRKNYLS(SEQ ID NO: 149) Consensus* LC-CDR1 KSSQSLLXXRTRKNYLX (SEQ ID NO: 212)wherein X at position 8 is N, I, Q or A; wherein X at position 9is S or T; and wherein X at position 17 is A or S 4D5 LC-CDR2 WASTRES(SEQ ID NO: 144) 1F3 LC-CDR2 WASTRES (SEQ ID NO: 144) 4B6 LC-CDR2WASTRES (SEQ ID NO: 144) Consensus LC-CDR2 WASTRES (SEQ ID NO: 144) 4D5LC-CDR3 KQSYNLYT (SEQ ID NO: 146) 1F3 LC-CDR3 KQSYNLYT (SEQ ID NO: 146)4B6 LC-CDR3 KQSYNLYT (SEQ ID NO: 146) Consensus LC-CDR3 KQSYNLYT(SEQ ID NO: 146) *Note: CDR-L1 consensus includes variants generated asdescribed in Example 19.

TABLE 22 Consensus Sequences for Group IB HC CDRs: Antibody RegionSequence 10D12 HC-CDR1 SYGMS (SEQ ID NO: 72) 35C1 HC-CDR1 SYGIT(SEQ ID NO: 79) Consensus HC-CDR1 SYGXX (SEQ ID NO: 213)wherein X at position 4 is M or I; and wherein X at position 5 is S or T10D12 HC-CDR2 WINTYSGVPTYADDFKG (SEQ ID NO: 74) 35C1 HC-CDR2WINTYSGVPTYADDFKG (SEQ ID NO: 74) Consensus HC-CDR2 WINTYSGVPTYADDFKG(SEQ ID NO: 74) 10D12 HC-CDR3 GGEAMDY (SEQ ID NO: 76) 35C1 HC-CDR3GGDALDY (SEQ ID NO: 82) Consensus HC-CDR3 GGXAXDY (SEQ ID NO: 214)wherein X at position 3 is E or D; and wherein X at position 5 is M or L

TABLE 23 Consensus Sequences for Group IB LC CDRs: Antibody RegionSequence 10D12 LC-CDR1 KSSQSLLDSDGKTYLN (SEQ ID NO: 153) 10D12-DELC-CDR1 KSSQSLLDSEGKTYLN (SEQ ID NO: 261) 10D12-DA LC-CDR1KSSQSLLDSAGKTYLN (SEQ ID NO: 262) 10D12-GA LC-CDR1 KSSQSLLDSDAKTYLN(SEQ ID NO: 263) 35C1 LC-CDR1 KSSQSLLDSDGKTYLS (SEQ ID NO: 159)Consensus* LC-CDR1 KSSQSLLDSXXKTYLX (SEQ ID NO: 215)Wherein X at position 10 is D, E or A; Wherein X at position 11is G or A; and wherein X at position 16 is N or S 10D12 LC-CDR2 LVSKLDS(SEQ ID NO: 155) 35C1 LC-CDR2 LVSKLDS (SEQ ID NO: 155) Consensus LC-CDR2LVSKLDS (SEQ ID NO: 155) 10D12 LC-CDR3 WQGTHFPWT (SEQ ID NO: 157) 35C1LC-CDR3 WQGTHFPYT (SEQ ID NO: 160) Consensus LC-CDR3 WQGTHFPXT(SEQ ID NO: 216) Wherein X at position 8 is W or Y *Note: CDR-L1consensus includes variants generated as described in Example 19.

DNA encoding mouse mAb heavy and light chains:

SEQ ID NO: 217: DNA encoding 4D5 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACAACCGACGATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGATAGAACTAAGTACAATGACAAGTTCAAGGACAAGGCCACATTGACTGTAGACACATCTTCCAACACAGCGTACATGGACCTCCACAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTTCAAGCCTCGAGGATACTTACTGGGGCCAAGGGACTCTGGTCGCTGTCTCTTCA SEQ ID NO: 218:DNA encoding 1F3 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGGAGTATTAAATATAATGAGAAATTCACGGACAAGGCCACATTGACAGTTGACGTATCCTCCAGCACAGCGTACATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTTCAGGTGTCGAGGATTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA SEQ ID NO: 219:DNA encoding 4B6 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAATTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGGTGAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGTACTACTAAGTACAATGAGGAGTTCACGGACAAGGCCACATTGACTGTTGACGTATCCTCCAGCACAGCGTTCATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTTCAAGTGTCGAGGATTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA SEQ ID NO: 220:DNA encoding 1A10 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAACGATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGAGATGGTACTACTAAGTACAATGAGAAGTTCACGGACAAGGCCACATTGACTGTTGACGTATCCTCCAGCACAGCGTTCATGGAGCTCCACAGGCTGACATCTGAGGACTCTGCGGTCTATTTCTGTTCAAGTGTCGAGGATTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA SEQ ID NO: 221:DNA encoding 10D12 heavy chain variable region (parental)CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATATTTTCACAAGCTATGGAATGAGCTGGGTGAGACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGAACTCCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGCGCAAGAGGGGGCGAAGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO: 222:DNA encoding 35C1 heavy chain variable region (parental)CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGACGCCAGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATATCTTCACATCCTATGGAATTACCTGGGTGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACGTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGACTACATATTTCTGTACAAGAGGGGGTGATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO: 223:DNA encoding 13B1 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGCTTTCCTGCAAGGCTACTGGCTACACATTCACTGGCAAGTGGATAGAGTGGGTAAAACAGAGGCCTGGACATGGCCTAGAGTGGATTGGAGAGATTTTACCTGGAACTGGTAGTACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGACTCATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACAACTGAAGACTCTGCTATGTATTATTGTTTAAGATCCGAGGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA SEQ ID NO: 224:DNA encoding 1G4 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGCTTGCCTGCAAGGCTACTGGCTACACATTCACTGGCTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATGTTACCTGGAAGTGGTAGTACTCACTACAATGAGAAGTTCAAGGGTAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCGGCCTGACAACTGAGGACTCTGCCATCTATTACTGTGTAAGAAGCATAGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO: 225:DNA encoding 1E7 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGGCCTGAGCTGGCAAGGCCTTGGGCTTCAGTGAAGATATCCTGCCAGGCTTTCTACACCTTTTCCAGAAGGGTGCACTTTGCCATTAGGGATACCAACTACTGGATGCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATCGGGGCTATTTATCCTGGAAATGGTGATACTAGTTACAATCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCATCCGGTAGCCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO: 226:DNA encoding 2D7 heavy chain variable region (parental)GAGGTCCAGCTGCAACAATCTGGGCCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGGTATCCTGTAAGGCTTCTGGATACACGCTCACTGACTACTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATGTTAATCCTAACAATGATGGTACTACCTACAACCAGAAATTCAAGGGCAGGGCCACATTGACTGTAGACAAGTCTTCCAACACAGCCTCCATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTACTACTGTGCAATATGCCCCTTTTATTACCTCGGTAAAGGGACCCACTTTGACTACTGGGGCCAAGGCAC CTCTCTCACAGTCTCCTCASEQ ID NO: 227: DNA encoding 49C11 heavy chain variable region(parental) GAGGTCCAGCTGCAACAATCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGGGAAGATGTCCTGTAAGGCTTCTGGATACAAATTCACTGACTACTATATGATCTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTTATTAAAATTTATAACGGTGGTACGAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGCCATCTCTCTATGATTACGACCCTTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA SEQ ID NO: 228:DNA encoding 15D9 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGAACTGAGCTGATGAAGCCTGGGGCCTCAGTGAACCTTTCCTGCAAGGCTTCTGGCTACACATTCACTGCCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTACTACTAACTACAATGAGAACTTCAAGGACAGGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACAAGTGAGGACTCTGCCATCTATTACTGTGCAAGATCCTATTACTACGCTAGTAGATGGTTTGCTTTCTGGGGCCAAGGGACTCTGGTCAC TGTCTCTTCASEQ ID NO: 229: DNA encoding 2F5 heavy chain variable region (parental)GAGGTCCAGCTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGATATTTATCCTGGTAGTGGTAGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGAGATACTACGCTACGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCAC TGTCTCTTCASEQ ID NO: 230: DNA encoding 1B11 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACTTTCACTGACTACTATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGCAAGGATTTATCCTGGAAGTGGTAATACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGAAAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGAAATTACTACATTAGTAGTCCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCAC TGTCTCTTCASEQ ID NO: 231: DNA encoding 2F2 heavy chain variable region (parental)CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTAGTGACGCCTGGAGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACTACCTATCCTATAGAGTGGATGAAACAGAATCATGGAAAGAGCCTAGAGTGGATTGGAAATTTTCATCCTTACAATGATGATACTAAGTACAATGAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGAAAAATCCTCTAACACAGTCTACTTGGAGCTCAGCCGATTAACATCTGATGACTCTGCTGTTTATTTCTGTGCAAGGAGGGTCTACTATAGTTACTTCTGGTTTGGTTACTGGGGCCACGGGACTCTGGTCAC TGTCTCTTCASEQ ID NO: 232: DNA encoding 11B6 heavy chain variable region (Parental)CAGGTGCAGCTGAAGCAGTCTGGGGCTGAGCTAGTGAAACCTGGAGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACTACCTATCCTATAGAGTGGATGAAGCAGAATCATGGGAAGAGCCTAGAGTGGATTGGAAATTTTCATCCTTACAATGGTGATTCTAAGTACAATGAAAAGTTCAAGGGCAAGGCCACCTTGACTGTAGAAAAATCCTCTAGCACAGTCTACTTAGAACTCAGCCGATTACCATCTGCTGACTCTGCTATTTATTACTGTGCAAGGAGGCACTACGCTGCTAGTCCCTGGTTTGCTCACTGGGGCCAAGGGACTCTGGTCAC TGTCTCTTCA

DNA encoding light chain variable region (mouse mAbs):

SEQ ID NO: 233: DNA encoding 4D5 light chain variable region (parental)GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGACCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCTCTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG SEQ ID NO: 234:DNA encoding 1F3 light chain variable region (parental)GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAGGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCATCAGTAGAACCCGAAAGAACTATTTGTCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTACAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGCGGGGGGACCAAGCTGGAAATAAAACGG SEQ ID NO: 235:DNA encoding 4B6/1A10 light chain variable region (parental)GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCATCAGTAGAACCCGAAAGAACTATTTGTCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTATTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTACAGGCTGAAGACCTGGCAGTTTATTACTGCAAACAATCTTATAATCTGTACACGTTCGGCGGGGGGACCAAGCTGGAAATCAAACGG SEQ ID NO: 236:DNA encoding 10D12 light chain variable region (parental)GATGTTTTGATGACCCAAACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG SEQ ID NO: 237:DNA encoding 35C1 light chain variable region (parental)GATATTGTGATGACGCAGGCTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAGTTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG SEQ ID NO: 238:DNA encoding 13B1 light chain variable region (parental)GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATATTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG SEQ ID NO: 239:DNA encoding 1G4 light chain variable region (parental)GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGAACAAGCCTCCATCTCTTGCAGATCAAGTCAGAGCCTTGTACAAAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG SEQ ID NO: 240:DNA encoding 1E7 light chain variable region (parental)GACATCCAGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG SEQ ID NO: 241:DNA encoding 2D7 light chain variable region (parental)GATATCCAGATGACACAGACTCCAGCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGTAGGGCAAGTCAGGACATTAGCAATTTTTTAAACTGGTATCAACAGAAACCGAATGGAACTGTTAAACTCCTAGTCTTCTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAGCAGAGCATTCTCTCACCATTAGCAACCTGGAGCAGGAAGATGTTGCCACTTACTTTTGCCAACAGGGTTTTACGCTTCCGTGGACGTTCGGTGGGGGCACCAAGGTGGAAATCAAACGG SEQ ID NO: 242:DNA encoding 49C11 light chain variable region (parental)GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCTTCTCTTGCAGATCTAGTCAGAGCCTTATACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG SEQ ID NO: 243:DNA encoding 15D9 light chain variable region (parental)GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAATAGGAGACAGGGTCAGCGTCACCTGCAGGGCCAGTCAGAATGTGGGTCCCAATTTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGATTCAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGGTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG SEQ ID NO: 244:DNA encoding 2F5 light chain variable region (parental)GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTCCTCGGCATCCAATCGGTACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAATATGCAGTCTGAAGACGTGGCAGATTATTTCTGCCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG SEQ ID NO: 245:DNA encoding 1B11 light chain variable region (parental)GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACTTCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTCCTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGACTATTTCTGTCAGCAATATAACCGCTATCCTCTCACGTTCGGTGCTGGGACCAAACTGGAGCTGAAACGG SEQ ID NO: 246:DNA encoding 2F2 light chain variable region (parental)GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAACGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTCATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGCGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACCTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCTCGAGCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG SEQ ID NO: 247:DNA encoding 11B6 light chain variable region (parental)GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAACGTCACCTGCAAGGCCAGTCAGAATGTGGGTCCTACTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTAATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCACTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG SEQ ID NO: 310:human IgG4 constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 311:human IgG4 constant region with S228P mutationASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 312:human IgG4 constant region with S228P mutation and and also amutation (Xtend) that promotes FcRn interations at low pHASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK SEQ ID NO: 313: human IgK constant regionTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 16

This Example describes functional characterization of recombinantpurified high affinity MASP-3 inhibitory antibodies in several in vitroassays.

Methods:

The recombinant MASP-3 mAbs generated as described in Examples 11 and 14were characterized for (i) binding to human MASP-3 and other species'MASP-3; (ii) the ability to inhibit cleavage of an artificial substrate;(iii) the capacity to inhibit pro-factor D to factor D cleavage; (iv)inhibition of complement deposition in human serum and (v) inhibition ofrabbit erythrocyte lysis in human serum as follows:

1. Assays to Determine Binding to Human and Mouse MASP-3

ELISA Assays:

MASP-3 Binding Assay with Purified Recombinant MASP-3 mAbs:

Human MASP-3:

A sandwich ELISA assay was carried out to measure binding of 16 purifiedrecombinant MASP-3 antibodies to human MASP-3 (CCP1-CCP2-SP fragment) asfollows. An ELISA plate was coated in carbonate/bi-carbonate bufferovernight at 4° C. with capture antibody αM3-259 at 4 μg/mL. αM3-259 isa high avidity recombinant, chimeric chicken-human MASP-3 mAb fromchickens immunized with the CCP1-CCP2-SP region of human MASP-3. Domainmapping studies revealed that αM3-259 binds the CCP1-CCP2 region ofMASP-3 from multiple species, including human, cynomolgus monkey, mouse,rat and dog. As shown in FIG. 51C, αM3-259 also binds to MASP-1.

The plate was subsequently blocked with 1% BSA/PBS, washed in PBS andthen incubated for one hour at room temperature with MASP-3 CCP1-CCP2-SP(2 μg/mL). The plate was then washed (PBS-T, 0.05%) and the candidateMASP-3 antibodies were added followed by incubation for one hour at roomtemperature. The plate was washed (PBS-T, 0.05%) and a detectionantibody was added (mouse anti-human kappa-HRP, SouthernBiotech#9230-05) for one hour at room temperature. After another wash (PBS-T,0.05%) the plate was developed (5 minutes) with OPT EIA TMB (BDBiosciences #555214). Absorbance reading at A450 was measured using theSpectramax M5e plate reader.

Results:

FIG. 51A and FIG. 51B graphically illustrate the avidities of MASP-3mAbs (purified recombinant) for human MASP-3 (CCP1-CCP2-SP). As shown inFIG. 51A, FIG. 51B, and Table 24, the MASP-3 mAbs have high avidity forhuman MASP-3, ranging from 0.241 nM to 0.023 nM. These values are 10 to100-fold lower than those reported for the previously described MASP-3mAbs (see Example 7 herein, also published as Example 15 inWO2013/192240).

MASP-3 mAb Binding Specificity:

To determine the specificity of the high affinity MASP-3 mabs forMASP-3, binding experiments were carried out to measure binding of 16purified recombinant MASP-3 antibodies to human MASP-1 and to humanMASP-2. Binding was determined as described for the MASP-3 bindingELISA, except that recombinant MASP-1A (S646A, CCP1-CCP2-SP fragment)and MASP-2 (CCP1-CCP2-SP fragment) were immobilized directly on theplate.

Results:

FIG. 51C graphically illustrates the results of a binding experiment inwhich representative purified recombinant high affinity human MASP-3inhibitory antibodies are shown to be selective for binding to MASP-3and do not bind to human MASP-1.

FIG. 51D graphically illustrates the results of a binding experiment inwhich representative purified recombinant high affinity human MASP-3inhibitory antibodies are shown to be selective for binding to MASP-3and do not bind to human MASP-2.

Mouse MASP-3:

Binding of the MASP-3 mAbs to mouse MASP-3 was measured as describedabove for human MASP-3 except that recombinant, full-length mouse MASP-3(SEQ ID NO:3) was captured on the plate with αM3-259. The negativecontrol mAb used in both experiments was mAb77, a recombinant, chimericchicken-human mAb obtained from the same immunized chickens as αM3-259,however, mAb 77 does not bind mouse MASP-3.

Results:

FIG. 52 graphically illustrates the avidities of representative MASP-3mAbs (purified recombinant) for mouse full length MASP-3. As shown inFIG. 52, most of the MASP-3 mAbs tested also have high avidity for mouseMASP-3.

The avidity values (EC₅₀) of the 16 recombinant chimeric MASP-3 mAbs forhuman and mouse MASP-3 are summarized in TABLE 24.

TABLE 24 Binding Avidity of MASP-3 mAbs for human and mouse MASP-3(FIGS. 51A, 51B and 52) Human MASP-3 Mouse MASP-3 Antigen used(CCP1-CCP2-SP) (full length) to generate Binding Avidity Binding AvidityAntibody clone mAb (EC₅₀ nM) (EC₅₀ nM) 1A10* SP 0.241 0.15 1B11 SP 0.0591.10 1E7 SP 0.112 117.00 1F3 SP 0.236 0.111 1G4 SP 0.177 3.70 2D7 SP0.122 NA 2F2 SP 0.057 0.105 2F5 SP 0.073 0.102 4B6 SP 0.211 0.188 4D5 SP0.058 0.098 10D12 CCP1-CCP2-SP 0.089 0.081 11B6 CCP1-CCP2-SP 0.060 0.06613B1 CCP1-CCP2-SP 0.059 0.035 15D9 CCP1-CCP2-SP 0.074 0.092 35C1CCP1-CCP2-SP 0.091 0.209 49C11 CCP1-CCP2-SP 0.069 0.064

Three of the MASP-3 mAbs-13B1, 10D12 and 4D5—were also tested forbinding to recombinant cynomolgus monkey, dog, and rat MASP-3. Theseresults are summarized below in Table 25.

TABLE 25 Summary of MASP-3 mAb Cross-Species Binding Experiments Speciesof MASP-3 Ranking of Fab Binding Human 13B1 (pM) ≈ 10D12 (pM) ≈ 4D5 (pM)Cynomolgus monkey 13B1 (pM) ≈ 4D5 (pM) > 10D12 (pM) Dog 13B1 (pM) >10D12 (pM) >> 4D5 (nM) Rat 13B1 (pM) ≈ 10D12 (pM) >> 4D5 (nM) Mouse10D12 (pM) > 13B1 (pM) >> 4D5 (nM)

As shown in TABLE 25, MASP-3 mAbs 13B1, 10D12 and 4D5 bind to all fivespecies of MASP-3 tested (human, mouse, rat, dog and cynomolgus monkey).While these mAbs bind to human with high avidity (<500 pM), they bind toother species of MASP-3 with varying avidities.

2. Fluorogenic Tripeptide Cleavage Assay

Background/Rationale:

In addition to its known natural substrates (Iwaki et al., J. Immunol.187:3751, 2011; Cortesio and Jiang, Arch. Biochem. Biophys. 449:164-170,2006), MASP-3 has been shown to hydrolyze various tripeptide substrates(Cortesio and Jiang, Ibid.). As very small substrates, these moleculescan be used to map the catalytic site of the protease. Inhibition oftri-peptide cleavage is an indication that an inhibitory agent, such asan antibody, either directly blocks access of the small substrate to thecatalytic site or causes a conformational shift in the SP domain thatsimilarly denies access. As such, the antibody can also be expected toblock catalysis of the large natural substrates by interfering with theactive site of the enzyme. Functionally, this would most closelyapproximate the MASP-3 null mouse or 3MC patient (deficient in MASP-3).

Methods:

Titrations of the recombinant mAbs (3-fold dilution from 666 nM to 0.91nM) were incubated with MASP-3 CCP1-CCP2-SP (197 nM) for 15 minutes atroom temperature. Tri-peptide substrate BOC-V-P-R-AMC(t-Butyloxycarbonyl-Val-Pro-Arg-7-Amino-4-methylcoumarin) (R&D Systems,Cat. No. ES011) was added at a final concentration of 0.2 mM. Hydrolysisof the Arg-AMC amide bond releases AMC, a highly fluorescent group.Excitation 380nm/emission 460nm kinetic values were recorded every 5minutes at 37° C. for 70 minutes using the Spectramax M5e fluorescenceplate reader.

Results:

FIG. 53 graphically illustrates the results of the assay measuringinhibition of MASP-3-dependent fluorogenic tripeptide cleavage with theMASP-3 monoclonal antibodies. As shown in FIG. 53, the MASP-3 mAbstested fall into three distinct groups:

-   -   1. MASP-3 mAbs that are strong inhibitors of peptide cleavage by        MASP-3: 1A10 (29.77 nM), 1G4 (29.64 nM), 1F3 (32.99 nM), 4B6        (26.03 nM), 4D5 (27.54 nM), 10D12 (30.94 nM) and 13B1 (30.13        nM).    -   2. MASP-3 mAbs that are weak or very weak inhibitors of peptide        cleavage by MASP-3: 15D9, 11B6, 2F5, 1E7 and 2D7    -   3. MASP-3 mAbs that are neutral or appear to stimulate peptide        cleavage by MASP-3: 1B11; 2F2; 77 (control mAb)

3. Inhibition of Pro-Factor D to Factor D Cleavage

Methods:

Active, recombinant human MASP-3 protein (240 ng per reaction) waspre-incubated with representative MASP-3 mAbs and a control mAb (whichbinds to MASP-1 but not to MASP-3) in GVB++buffer with a total volume of9 μL at room temperature for 15 minutes. 70 ng of pro-factor D with anN-terminal Strep-tag II epitope tag (ST-pro-factor D-His) was then addedto each tube to make the final volume per reaction to 10 μL. Thereactions were incubated in a thermocycler at 37° C. for 6 hours. Onetenth from each reaction was then electrophoresed on a 12% Bis-Tris gelto resolve pro-factor D and active factor D cleavage product. Theresolved proteins were transferred to a PVDF membrane and analyzed usingWestern blot by detection with a biotinylated factor D antibody (R&DSystems).

Results:

FIG. 54 shows a Western blot analysis demonstrating the ability ofrepresentative MASP-3 mAbs to block recombinant MASP-3-mediated cleavageof pro-CFD to CFD in an in vitro assay. As shown in FIG. 54,representative high affinity MASP-3 inhibitory mAbs 13B1, 4B6, 1G4, 2D7,10D12, 1A10, 4D5, 1E7, and 1F3 mouse-human chimeric mAbs showed partialto full inhibition of the pro-CFD cleavage in this assay.

4. Factor Bb Deposition on Zymosan Assay

Methods:

Varying concentrations of MASP-3 mAbs were added to 10% CFD-depletedhuman serum (Complement Technology A336) and GVB+Mg/EGTA (20 nM) andincubated for 30 minutes on ice prior to the addition of recombinantST-pro-factor D-His (2 μg/mL final) and zymosan (0.1 mg/mL final). Thezymosan particles function as an activating surface for complementdeposition. The mixtures were incubated at 37° C. and the APC activitywas measured by the flow cytometric detection of complement factor Bb(Quidel antibody A252) on the surface of the zymosan particles.

Results:

FIG. 55A graphically illustrates the level of factor Bb deposition onzymosan particles (determined by flow cytometric detection measured inMFI units) in the presence of varying concentrations of MASP-3 mAbs 1F3,1G4, 2D7 and 4B6 in factor D-depleted human serum at 37° C. for 70minutes.

FIG. 55B graphically illustrates the level of factor Bb deposition onzymosan particles (determined by flow cytometric detection measured inMFI units) in the presence of varying concentrations of MASP-3 mAbs 4D5,10D12 and 13B1 in CFD-depleted human serum at 37° C. for 70 minutes.

The results shown in FIGS. 55A and 55B are summarized below in TABLE 26.

TABLE 26 Inhibition of Factor Bb deposition on zymosan by MASP-3 mAbs(FIG. 55A and FIG. 55B) Inhibition of Factor Bb Deposition on ZymosanAntibody (IC₅₀ nM) 1F3 0.1 1G4 1.1 2D7 3.5 4B6 0.2 4D5 0.4 10D12 0.513B1 0.3

As shown in FIG. 55A, FIG. 55B and TABLE 26, the MASP-3 mAbs show potentinhibition of the APC in human serum, with IC₅₀ values ranging from 0.1nM to 3.5 nM. These results demonstrate that MASP-3 plays a key role inAPC activation in an in vitro model in human serum, and furtherdemonstrate that MASP-3 inhibitory antibodies are potent inhibitors ofthe APC.

5. Assay to Measure the Ability of Representative MASP-3 mAbs to InhibitRabbit Erythrocyte Lysis

Methods:

To monitor the inhibition of the APC in another experimental context, weevaluated the ability of representative MASP-3 mAbs to block the lysisof rabbit erythrocytes in human serum. Varying concentrations of MASP-3mAbs were added to 10% factor D-depleted human serum and GVB+Mg/EGTA (20nM) and incubated for 30 minutes on ice prior to the addition ofrecombinant ST-pro-factor B-His (2 μg/mL final) and erythrocytes(2.5×10⁸ cells/mL final). The mixtures were incubated at 37° C. for 70minutes and APC-mediated hemolysis was measured by diluting thereactions and measuring the absorbance (A405), which indicates levels offree hemoglobin.

Results:

FIG. 56A graphically illustrates the level of inhibition of rabbiterythrocyte lysis in the presence of varying concentrations of MASP-3mAbs 1A10, 1F3, 4B6, 4D5, 1G4 and 2F2 in CFD-depleted human serum. FIG.56B graphically illustrates the level of inhibition of rabbiterythrocyte lysis in the presence of varying concentrations of MASP-3mAbs 1B11, 1E7, 1G4, 2D7 and 2F5 in CFD-depleted human serum. Theresults are summarized in TABLE 27.

TABLE 27 Inhibition of Rabbit Erythrocyte Lysis by MASP-3 mAbs (FIG. 56Aand FIG. 56B) Inhibition of Rabbit Erythrocyte Antibody Lysis (IC₅₀ nM)1A10 0.2 1F3 0.2 4B6 0.2 4D5 0.1 1G4 2.7 2F2 0.8 1B11 NA 1E7 NA 2D7 5.42F5 0.9

As shown in FIG. 56A, FIG. 56B and TABLE 27, the MASP-3 mAbs showinhibition of the APC-driven hemolysis of rabbit erythrocytes, with IC50values ranging from 0.1 nM to 5.4 nM. These results corroborate theobservations of the MASP-3 antibodies in the zymosan assay, and furtherdemonstrate that MASP-3 inhibitory antibodies are potent inhibitors ofthe APC.

6. Inhibition of Pro-Factor D Cleavage in 3MC Patient Serum

Methods:

A representative recombinant MASP-3 mAb (4D5) was tested for the abilityto block recombinant MASP-3 cleavage (and activation) of pro-factor Doriginating from normal human serum and serum from 3MC Patient B (“PatB”), an individual who has no detectable MASP-3 in the serum andmanifests a deficiency in the APC.

Normal human serum and Patient B serum (10% final) and GVB +Mg/EGTA (30nM) were incubated with no enzyme or with active recombinant MASP-3(rMASP-3; 0.5 μg/mL), inactive rMASP-3, or active rMASP-3 plus MASP-3mAb 4D5 (500 nM final) on ice for 1 hour. Zymosan (0.1 mg/mL final) wasadded, and the mixtures were incubasted at 37° C. After 2 hours, thesamples were centrifuged and the supernatants were collected. Thesamples were immunoprecipitated with goat antibody raised against humanFactor D (R&D Systems AF1824), heat denatured and treated withPeptide-N-Glycosidase (New England Biolabs P0704L). The captured anddeglycosylated proteins were resolved with SDS-PAGE and the gels wereelectroblotted for Western blot analysis with a biotinylated anti-CFD(R&D Systems BAF1824) and High Sensitivity Streptavidin-HRP (ThermoFischer Scientific 21130).

Results:

FIG. 57 shows a Western blot analyzing the level of pro-factor D andfactor Din 3MC Patient B serum in the presence active rMASP-3, inactiverMASP-3, and active rMASP-3 plus mAb 4D5. As shown in FIG. 57, normalhuman serum contains predominately the mature form, while Patient Bserum principally contains the zymogen form of factor D. As furthershown in FIG. 57, active rMASP-3 in the presence of zymosan causescleavage of pro-factor D in Patient 3 serum, while the inactive(zymogen) form of MASP-3 does not. Finally, as shown in FIG. 57, theMASP-3 mAb 4D5 blocks cleavage of pro-factor D in Patient 3 serum in thepresence of active rMASP-3. These results further demonstrate the roleof MASP-3 in the cleavage of pro-factor D in the activation of the APC,and demonstrate that a MASP-3 inhibitory mAb is capable of blockingMASP-3 mediated pro-factor D cleavage and thereby blocking the APC.

Example 17

Analysis of representative MASP-3 inhibitory mAbs 10D12 and 13B1 for theability to inhibit the APC In Vvivo.

1. Inhibition of the APC by mAb M3-1 (13B1) and 10D12 In Vivo:

Methods:

In order to determine the efficacy of MASP-3 mAb 13B1 (M3-1) and 10D12for inhibiting the APC in vivo, a group of mice (n =4) received a singleintravenous tail vein injection of 10 mg/kg mAb 13B1 and a second groupof mice (n=4) received a single intravenous tail vein injection of 10mg/kg mAb 10D12. Blood collected from the animals was used to prepareserum, providing a matrix for the flow cytometric assessment of APCactivity in an ex vivo assay measuring the level of C3 (also C3b andiC3b, Dako F020102-2) deposition on zymosan particles. Serum preparedfrom blood harvested at a pre-dose timepoint and multiple post-dose timepoints (96 hrs, 1 week, and 2 weeks) was diluted to 7.5% and zymosanparticles (0.1 mg/mL final) were added to induce the APC.Antibody-treated mice were compared to a group of control mice (n=4)that were given a single intravenous dose of vehicle.

Results:

FIG. 58 graphically illustrates the level of C3 deposition on zymosanparticles at various time points after a single dose of mAb M3-1 (13B1),mAb 10D12, or vehicle in wild-type mice. As shown in FIG. 58, in thepre-dose time point the three conditions show comparable levels of APCactivity. At 96 hours and the two later time points, both mAb-treatedgroups show near-complete ablation of systemic APC activity, while theAPC activity of the vehicle-treated group remains unabated.

These results demonstrate that MASP-3 mAb M3-1 (13B1) and mAb 10D12 arepotent inhibitors of the APC in vivo in mouse.

2. Status of Factor B in mice treated with MASP-3 mAb 10D12

Methods:

During the conversion of Factor B zymogen to an active proteolyticenzyme, Factor B is cleaved into the Ba (˜30 kDa) and Bb (˜60 kDa)fragments by Factor D. The status of the Ba fragment in mouse serumobtained from mice treated with the MASP-3 mAb 10D12 was determined asfollows.

Mice (n=4) were given two intravenous tail vein injections of 10 mg/kgmAb 10D12.The treatments occurred seven days apart and blood wascollected from the animals three days after the second injection. Asecond set of four mice received a single intravenous dose of vehicle(PBS). The blood collected from both groups was used to prepare serum,providing a matrix for complement activation. Zymosan particles (0.1mg/mL final) were added to diluted serum (7.5% final) and incubated for35 minutes at 37° C.

Results:

As a measure of APC activation, FIG. 59 shows a Western blot analyzingthe status of the Ba fragment in mouse serum obtained from mice treatedwith mAb 10D12 or PBS and stimulated with zymosan. Each lane in FIG. 59represents a different mouse, and the lanes alternate to show serum froma representative vehicle mouse adjacent to a MASP-3 mAb-treated mousefor the purposes of comparison. Two control conditions, from micetreated with vehicle or mAb 10D12 are shown in lanes 1 and 2,respectively (starting from the left side of the blot) asrepresentatives of the basal level of Ba present in the serum samples inthe absence of zymosan. Lanes 3 to 10 all show the level of Ba fragmentpresent after incubation with zymosan. In all cases, the MASP-3mAb-treated mice demonstrate a reduced level of the Ba fragment incomparison to the vehicle-treated animals.

3. Serum from Mice Treated with mAb 10D12 Inhibits Hemolysis

Methods:

As another measure of APC inhibition by MASP-3 inhibitory antibodies, weevaluated the ability of the MASP-3 antibodies to block the lysis ofrabbit erythrocytes in serum from mice treated with representativeMASP-3 mAb 10D12 as compared to serum from vehicle control treated mice.

Mice (n=4/group) were given three intravenous tail vein injections ofvehicle control (PBS), 10 mg/kg MASP-3 mAb 10D12, or 25 mg/kg MASP-3 mAb10D12. The treatments occurred seven days apart from one another andblood was collected from the animals three days after the thirdinjection. The blood was used to prepare serum, providing a matrix forhemolysis reactions.

Erythrocytes (2.5×10⁸ cells/mL final) were added to 20% pooled serumfrom four mice in GVB+Mg/EGTA (20 nM). The mixtures were incubated at37° C. and APC-mediated hemolysis was measured by diluting the reactionsand measuring the absorbance (A405).

Results:

FIG. 60 graphically illustrates the level of inhibition of hemolysis by20% serum from mice treated with MASP-3 mAb 10D12 (10 mg/kg or 25 mg/kg)or vehicle control treated mice. As shown in FIG. 60, serum from micetreated with MASP-3 mAb 10D12 at both 10 mg/kg and 25 mg/kg demonstratedless overall hemolysis during the 1 hour test period as compared tovehicle-treated mice.

Overall Summary of Results:

As described in this Example, representative high affinity MASP-3inhibitory mAbs 13B1 and 10D12 inhibit the APC in vivo. As described inExample 12, it was determined that MASP-3 monoclonal antibody 13B1 (alsoreferred to as mAb M3-1) provides a clear benefit to survival of redblood cells lacking Crry in a mouse model associated with paroxysmalnocturnal hemogloinuria (PNH). As described in Example 13, it wasdetermined that MASP-3 mAb M3-1 reduced the incidence and severity ofclinical arthritis scores in a dose-dependent fashion.

Example 18

This Example describes the results of epitope binding analysis of highpotency MASP-3 inhibitory mAbs.

1. Competition Binding Analysis

Methods:

96 well ELISA assay plates were coated with the capture antibody,αM3-259, an IgG4 isotype mAb which has been shown to bind the CCP1-CCP2region of MASP-1 and MASP-3. The full-length human MASP-3 protein wasimmobilized on the plate via capture antibody αM3-259. In separate,non-coated wells, a 2-fold dilution series of one test MASP-3 mAb of anIgG4 isotype was mixed with a constant concentration of another testMASP-3 antibody of an IgG1 isotype. The mixture was added to the coatedwells and allowed to bind to the captured MASP-3. Potential competitionbetween the two antibodies was determined by the detection of the IgG1isoform using an HRP-conjugated antibody against the human IgG1 hingeregion (Southern Biotech 9052-05), and a TMB substrate reagent set (BDBiosciences 555214).

Results:

FIGS. 61A-61E graphically illustrate the results of the competitionbinding analysis.

FIG. 61A graphically illustrates the results of the competition bindinganalysis to identify MASP-3 mAbs (IgG4) that block the interactionbetween mAb 4D5 (IgG1) and human MASP-3.

FIG. 61B graphically illustrates the results of the competition bindinganalysis to identify MASP-3 mAbs (IgG4) that block the interactionbetween mAb 10D12 (IgG1) and human MASP-3.

FIG. 61C graphically illustrates the results of the competition bindinganalysis to identify MASP-3 mAbs (IgG4) that block the interactionbetween mAb 13B1 (IgG1) and human MASP-3.

FIG. 61D graphically illustrates the results of the competition bindinganalysis to identify MASP-3 mAbs (IgG4) that block the interactionbetween mAb 1F3 (IgG1) and human MASP-3.

FIG. 61E graphically illustrates the results of the competition bindinganalysis to identify MASP-3 mAbs (IgG4) that block the interactionbetween mAb 1G4 (IgG1) and human MASP-3.

The data from FIGS. 61A to 61E is summarized below in TABLE 28.

These data indicate that MASP-3 mAbs 4D5, 10D12, 13B1, 1A10, 1F3 and 1G4share a common epitope or overlapping epitopes on human MASP-3.Surprisingly, 1G4 has a very limited capacity to block the binding ofthe other five mAbs to MASP-3, but those mAbs almost completely blockthe binding of 1G4 itself to MASP-3.

2. Analysis of mAb Binding to Peptides Representing Linear andDiscontinuous

MASP-3 Epitopes

Methods:

Fourteen of the 16 MASP-3 mAbs were evaluated by Pepscan to identify theregions of MASP-3 to which they bind. To reconstruct both linear andpotential discontinuous epitopes of the target molecule, a library ofpeptides was synthesized corresponding to amino acid residues 299 to 728of SEQ ID NO:2 (human MASP-3). Amino acid residues 1-298 of MASP-3 werenot present in the immunogen and were not included in this analysis.

Pepscan epitope analsysis included use of the CLIPS technology, whichstructurally fixes peptides into defined three-dimensional structures(see Timmerman et al., J Mol Recog. 20:283-299, 2007 and Langedijk etal., Analytical Biochemistry 417:149-155, 2011). The binding of eachantibody to each of the synthesized peptides was tested in aPepscan-based ELISA.

Results:

The peptide binding results from Pepscan for each antibody analyzed isdescribed below and summarized in TABLE 4, TABLE 28 and FIGS. 62-67.

Antibodies 1F3, 4B6, 4D5 and 1A10 (Group IA)

When tested under moderate stringency conditions, antibodies 1F3, 4B6,4D5 and 1A10 bound discontinuous epitope mimics and also bound simpleconstrained and linear mimics. Data analysis demonstrates thatantibodies 1F3, 4B6, 4D5 and 1A10 all dominately recognize peptidestretch 498VLRSQRRDTTVI5o9 (SEQ ID NO:9) of MASP-3. This peptide liesimmediately adjacent to the active site histidine, H497. Data obtainedfor these antibodies with discontinuous mimics suggest that peptidestretches ₅₄₄DFNIQNYNHDIALVQ₅₅₈ (SEQ ID NO:11), ₆₃₉GNYSVTENMFC₆₄₉ (SEQID NO:13) and ₇₀₄VSNYVDWVWE₇₁₃ (SEQ ID NO:14) of MASP-3 also contributeto the binding. Peptide ₅₄₄DFNIQNYNHDIALVQ₅₅₈ (SEQ ID NO:11) containsthe active site aspartate (D553).

Antibody 10D12 (Group IB)

When tested under moderate stringency conditions, antibody 10D12 boundpeptides with core sequence ₄₉₈VLRSQRRDTTVI₅₀₉ (SEQ ID NO:9) of MASP-3,the sequence adjacent to the active site histidine, H497.

Antibody 13B1 (Group IC)

When tested under moderate stringency conditions antibody 13B1recognizes a discontinuous epitope comprising peptide stretches₄₉₄TAAHVLRSQRRDTTV₅₀₈ (SEQ ID NO:10) and ₆₂₆PHAECKTSYESRS₆₃₈ (SEQ IDNO:12) of MASP-3, where peptide stretch ₆₂₆PHAECKTSYESRS₆₃₈ (SEQ IDNO:12) appears to be the dominant part of the epitope as it can also bebound in simple constrained form. The peptide ₄₉₄TAAHVLRSQRRDTTV₅₀₈ (SEQID NO:10) includes the active site histidine, H497.

Antibody 1G4 (Group II)

When tested under low stringency conditions antibody 1G4 recognizes adiscontinuous epitope comprising peptide stretches ₄₅₄RNAEPGLFPWQ₄₆₄(SEQ ID NO:17), ₅₁₄EHVTVYLGLH₅₂₃ (SEQ ID NO:19) and ₆₆₇AFVIFDDLSQRW₆₇₈(SEQ ID NO:23) of MASP-3, where peptide stretch ₆₆₇AFVIFDDLSQRW₆₇₈ (SEQID NO:23) is the dominant part of the epitope. The dominant peptide lieswithin three amino acids of the active site serine, S664.

Antibodies 1E7 and 2D7 (Group IIIA)

When tested under high and low stringency conditions, respectively,antibodies 1E7 and 2D7 recognize a discontinuous epitope comprisingpeptide stretches ₄₅₄RNAEPGLFPWQ₄₆₄ (SEQ ID NO:17), ₅₁₄EHVTVYLGLH₅₂₃(SEQ ID NO:19) and ₆₆₇AFVIFDDLSQRW₆₇₈ (SEQ ID NO:23) of MASP-3, wherepeptide stretch ₆₆₇AFVIFDDLSQRW₆₇₈ (SEQ ID NO:23) is the dominant partof the epitope and which lies within three amino acids of the activesite serine, S664.

Antibodies 2F5 and 15D9 (Group IIIB)

When tested under low stringency conditions, antibodies 2F5 and 15D9dominantly recognize a discontinuous epitope comprising peptidestretches ₄₅₄RNAEPGLFPWQ₄₆₄ (SEQ ID NO:17), ₄₇₉KWFGSGALLSASWIL₄₉₃ (SEQID NO:18), ₅₆₂PVPLGPHVMP₅₇₁ (SEQ ID NO:20) and ₆₆₇AFVIFDDLSQRW₆₇₈ (SEQID NO:23) of MASP-3. Peptides ₄₇₉KWFGSGALLSASWIL₄₉₃ (SEQ ID NO:18) and₆₆₇AFVIFDDLSQRW₆₇₈ (SEQ ID NO:23) localize within four or three aminoacids of the active site residues H497 and 5664, respectively.

Antibody 1B11 (Group IIIC)

When tested under moderate stringency conditions, antibody 1B11recognizes a discontinuous epitope comprising peptide stretches₄₃₅ECGQPSRSLPSLV₄₄₇ (SEQ ID NO:16), ₄₅₄RNAEPGLFPWQ₄₆₄ (SEQ ID NO:17),₅₈₃APHMLGL₅₈₉ (SEQ ID NO:21) and ₆₁₄SDVLQYVKLP₆₂₃ (SEQ ID NO:22) ofMASP-3.

TABLE 28 Summary of Epitope Binding Analysis MASP-3Peptide Binding Fragments Peptide mAb Ref. (Epitopes) on human CompetesCleavage No./Group MASP-3 (w/leader) With Assay 4D5₄₉₈VLRSQRRDTTVI₅₀₉ (SIN: 9) 1F3, 1G4, 4D5, inhibits Group IA₅₄₄DFNIQNYNHDIALVQ₅₅₈ (SIN: 11) 10D12, 13B1 ₆₃₉GNYSVTENMFC₆₄₉ (SIN: 13)₇₀₄VSNYVDWVWE₇₁₃ (SIN: 14) 1F3 ₄₉₈VLRSQRRDTTVI₅₀₉ (SIN: 9)1F3, 1G4, 4D5, inhibits Group IA ₅₄₄DFNIQNYNHDIALVQ₅₅₈ (SIN:11)10D12, 13B1 ₆₃₉GNYSVTENMFC₆₄₉ (SIN: 13) ₇₀₄VSNYVDWVWE₇₁₃ (SIN: 14) 4B6₄₉₈VLRSQRRDTTVI₅₀₉ (SIN: 9) 1F3, 1G4, 4D5, inhibits Group IA₅₄₄DFNIQNYNHDIALVQ₅₅₈ (SIN: 11) 10D12, 13B1 ₆₃₉GNYSVTENMFC₆₄₉ (SIN: 13)₇₀₄VSNYVDWVWE₇₁₃ (SIN: 14) 1A10 ₄₉₈VLRSQRRDTTVI₅₀₉ (SIN: 9)1F3, 1G4, 4D5, inhibits Group IA ₅₄₄DFNIQNYNHDIALVQ₅₅₈ (SIN: 11)10D12, 13B1 ₆₃₉GNYSVTENMFC₆₄₉ (SIN: 13) ₇₀₄VSNYVDWVWE₇₁₃ (SIN: 14) 10D12₄₉₈VLRSQRRDTTVI₅₀₉ (SIN: 9) 1F3, 1G4, 4D5, inhibits Group IB 10D12, 13B113B1 ₄₉₄TAAHVLRSQRRDTTV₅₀₈ (SIN: 10) 1F3, 1G4, 4D5, inhibits Group IC₆₂₆PHAECKTSYESRS₆₃₈ (SIN: 12) 10D12, 13B1 Group I₄₉₈VLRSQRRDTTV₅₀₈ (SIN: 15) core sequence 1G4₄₅₄RNAEPGLFPWQ₄₆₄ (SIN: 17) 1F3, 1G4, 4D5, inhibits Group II-₅₁₄EHVTVYLGLH₅₂₃ (SIN: 19) 10D12, 13B1 cross₆₆₇AFVIFDDLSQRW₆₇₈ (SIN: 23) competes with Group I and III 1E7₄₅₄RNAEPGLFPWQ₄₆₄ (SIN: 17) 1G4 Weakly Group IIIA₅₁₄EHVTVYLGLH₅₂₃ (SIN: 19) inhibits ₆₆₇AFVIFDDLSQRW₆₇₈ (SIN: 23) 2D7₄₅₄RNAEPGLFPWQ₄₆₄ (SIN: 17) Weakly Group IIIA ₅₁₄EHVTVYLGLH₅₂₃ (SIN: 19)inhibits ₆₆₇AFVIFDDLSQRW₆₇₈ (SIN: 23) 2F5 ₄₅₄RNAEPGLFPWQ₄₆₄ (SIN: 17)No effect Group IIIB ₄₇₉KWFGSGALLSASWIL₄₉₃ (SIN 18)₅₆₂PVPLGPHVMP₅₇₁ (SIN: 20) ₆₆₇AFVIFDDLSQRW₆₇₈ (SIN: 23) 15D9₄₅₄RNAEPGLFPWQ₄₆₄ (SIN: 17) No effect Group IIIB₄₇₉KWFGSGALLSASWIL₄₉₃ (SIN 18) ₅₆₂PVPLGPHVMP₅₇₁ (SIN: 20)₆₆₇AFVIFDDLSQRW₆₇₈ (SIN: 23) 1B11 ₄₃₅ECGQPSRSLPSLV₄₄₇ (SIN: 16)stimulates Group IIIC ₄₅₄RNAEPGLFPWQ₄₆₄ (SIN: 17)₅₈₃APHMLGL₅₈₉ (SIN: 21) ₆₁₄SDVLQYVKLP₆₂₃ (SIN: 22) Core₄₅₄RNAEPGLFPWQ₄₆₄ (SIN: 17) sequence for Group II and Group III 2F2Binding epitope not determined 1F3, 4D5, 11B6, stimulates Group IV 2F211B6 Binding epitope not determined 1F3, 4D5, 11B6, No effect Group IV2F2

FIG. 62 provides a schematic diagram showing the regions of contact onhuman MASP-3 by the MASP-3 mAbs, as determined by Pepscan Analysis. Asshown in FIG. 62, all of the MASP-3 mAbs have regions of contact in thebeta chain containing the SP domain of MASP-3. One mAb, 1B11, also has aregion of contact between the CCP2 and SP domains in the alpha chain ofMASP-3.

FIGS. 63A to 67 show 3-D models illustrating the regions of contact ofthe high affinity MASP-3 mAbs on the CCP1/2/SP domains of human MASP-3,wherein the SP domain active site of MASP-3 is facing towards the frontand the catalytic triad is shown as side chains.

FIG. 63A shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAbs 1F3, 4D5 and 1A10, including aa residues 498-509(SEQ ID NO:9), aa residues 544-558 (SEQ ID NO:11), aa residues 639 to649 (SEQ ID NO:13) and aa residues 704 to 713 (SEQ ID NO:14).

FIG. 63B shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAb 10D12, including aa residues 498 to 509 (SEQ IDNO:9).

FIG. 64 shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAb 13B1, including aa residues 494 to 508 (SEQ IDNO:10) and aa residues 626 to 638 (SEQ ID NO: 12).

FIG. 65 shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAb 1B11, including aa residues 435 to 447 (SEQ IDNO:16), aa residues 454 to 464 (SEQ ID NO:17), aa residues 583 to 589(SEQ ID NO:21) and aa residues 614 to 623 (SEQ ID NO:22).

FIG. 66 shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAbs 1E7, 1G4 and 2D7, including aa residues 454 to 464(SEQ ID NO:17), aa residues 514 to 523 (SEQ ID NO:19) and aa residues667 to 678 (SEQ ID NO:23).

FIG. 67 shows the regions of contact between human MASP-3 and highaffinity MASP-3 mAbs 15D9 and 2F5, including aa residues 454 to 464 (SEQID NO:17), aa residues 479 to 493 (SEQ ID NO:18), aa residues 562 to 571(SEQ ID NO:20), and aa residues 667 to 678 (SEQ ID NO:23).

In summary, conclusive binding profiles were obtained for 12 of the 14antibodies. All 12 mapped antibodies recognized solvent exposed epitopeswithin the peptidase 51 domain. The close proximity of a number of theepitope determinants to residues for the active site catalytic triad(H497, D553, 5664) is consistent with a model in which the high affinityinhibitory MASP-3 mAbs block enzymatic activity by interfering with theenzyme-substrate interaction.

Example 19

This Example describes the humanization of representative MASP-3 mAbsand engineering of potential post-translational modification sites.

Methods:

1. Humanization of Representative High Affinity MASP-3 mAbs Methods:

To reduce immunogenicity risk, representative high affinity MASP-3inhibitory antibodies 4D5, 10D12 and 13B1 were humanized by aCDR-grafting method. CDRS of each MASP-3 antibody were grafted into theclosest consensus human framework sequences. Some of the Vernier zoneresidues were modified by Quickchange site-directed mutagenesis (AgilentTechnologies). The resulting humanized VH and VL regions weretransferred into pcDNA3.1-based human IgG1 or IgG4 and IgK expressioncontructs, and the recombinant antibodies were expressed and purified asdescribed above. Affinity of the humanized antibodies was determined byELISA using monovalent Fab fragments, and potency was assessed by C3deposition assay using intact IgG4 formats.

Results:

Amino acid sequences of representative humanized versions of the heavychain variable regions and light chain variable regions for mAbs 4D5,10D12 and 13B1 are provided below. The CDRs (Kabat) are underlined.

4D5: h4D5_VH-14 (SEQ ID NO: 248)QVQLVQSGAEVKKPGASVKVSCKASGYTFTTDDINWVRQAPGQGLEWIGWIYPRDDRTKYNDKFKDKATLTVDTSSNTAYMELSSLRSEDTAVYYCSSLE DTYWGQGTLVTVSSh4D5_VH-19 (SEQ ID NO: 249)QVQLVQSGAEVKKPGASVKVSCKASGYTFTTDDINWVRQAPGQGLEWIGWIYPRDDRTKYNDKFKDRATLTVDTSSNTAYMELSSLRSEDTAVYYCSSLE DTYWGQGTLVTVSSh4D5_VL-1 (SEQ ID NO: 250)DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNL YTFGQGTKVEIKR 10D12:h10D12_VH-45 (SEQ ID NO: 251)QIQLVQSGSELKKPGASVKVSCKASGYIFTSYGMSWVRQAPGKGLKWMGWINTYSGVPTYADDFKGRFVFSLDTSVRTPYLQISSLKAEDTAVYFCARGG EAMDYWGQGTLVTVSSh10D12_VH-49 (SEQ ID NO: 252)QIQLVQSGSELKKPGASVKVSCKASGYIFTSYGMSWVRQAPGKGLKWMGWINTYSGVPTYADDFKGRFVFSLDTSVRTPYLQISSLKAEDTATYFCARGG EAMDYWGQGTLVTVSSh10D12_VL-21 (SEQ ID NO: 253)DVLMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFP WTFGQGTKVEIKR 13B1h13B1_VH-9 (SEQ ID NO: 254)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGKWIEWVRQAPGQGLEWIGEILPGTGSTNYAQKFQGRATFTADSSTSTAYMELSSLRSEDTAVYYCLRSE DVWGQGTLVTVSSh13B1_VH-10 (SEQ ID NO: 255)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGKWIEWVRQAPGQGLEWIGEILPGTGSTNYNEKFKGRATFTADSSTSTAYMELSSLRSEDTAVYYCLRSE DVWGQGTLVTVSSh13B1_VL-1 (SEQ ID NO: 256)DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNI PTFGQGTKVEIKR

The affinity of representative humanized 4D5, 10D12 and 13B1 antibodiesfor human MASP-3 is shown below in TABLE 29.

TABLE 29 Binding of Representative humanized MASP-3 mAbs to MASP-3MASP-3 antibody clone Binding to human MASP-3 (Fab format) EC₅₀ (nM) 4D5Parental Fab 0.107 h4D5_14-1 Fab 0.085 (VH-14 and VL-1) h4D5_19-1 Fab0.079 (VH-19 and VL-1) 10D12 Parental Fab 0.108 h10D12_45-21 Fab 0.108(VH-45 and VL-21) h10D12_49-21 Fab 0.115 (VH-49 and VL-21) 13B1 ParentalFab 0.123 h13B1_9-1 Fab 0.101 (VH-9 and VL-1) h13B1_10-1 Fab 0.097(VH-10 and VL-1)

The percent identity of humanized framework sequences to those of humangermline framework sequences:

-   h4D5 VH-14=90%; h4D5 VH-19=91%; h4D5 VL-1=100%;-   h10D12 VH-45=92%; h10D12 VH-49=91%; h10D12 VL-21=93%;-   h13B1 VH-9=95%; h13B1 VH-10=94%; h13B1 VL-1=100% 2. Mutagenesis of    Representative MASP-3 mAbs to Remove Asn/Asp Modification Sites in    CDR-1 of the Light Chain Variable Region of 4D5, 10D12 and 13B1

Representative high affinity MASP-3 inhibitory mAbs 4D5, 10D12 and 13B1were analyzed for post-translational modification. Asparagine residueswith a succeeding Glycine, Serine, Histidine, Alanine or Asparagine(“NG”, “NS”, “NH”, “NA”, or “NN” motif) are often susceptible to thehydrolysis of the amide group of Asparagine side-chain, or“deamidation.” Aspartic acid residues with a succeeding Glycine orProline (“DG” or “DP” motif) are often susceptible to theinterconversion, or “isomerization.” Such modifications result in chargeheterogeneity and may affect antibody function if they occur in abinding interface. They also may increase risks of fragmentation,immunogeneticity and aggregation.

Potential post-translational modification motifs were identified inCDR-1 of the light chain variable regions of 4D5, 10D12 and 13B1.

4D5 and 13B1 contained one possible Asn deamidation site in CDR1 of thelight chain (shown as “NS” at positions 8 and 9 of SEQ ID NO:142underlined in TABLE 30 below. As further shown below in Table 30, 10D12contained one possible Asp isomerization site in CDR1 of the lightchain.

Variants of the humanized version of these MASP-3 mAbs were generated bysite-directed mutagenesis as shown in TABLE 30. The variants wereexpressed and purified as described above. Affinity was determined byELISA using monovalent Fab fragments, and potency was assessed by C3deposition assay using intact IgG4 formats as described above.

TABLE 30 Variants of CDR-L1 for 4D5, 10D12 and 13B1 Antibody RegionSequence 4D5 parent LC-CDR1 KSSQSLLNSRTRKNYLA (SEQ ID NO: 142)4D5-NQ mutant LC-CDR1 KSSQSLLQSRTRKNYLA (SEQ ID NO: 257) 4D5-NA mutantLC-CDR1 KSSQSLLASRTRKNYLA (SEQ ID NO: 258) 4D5-ST mutant LC-CDR1KSSQSLLNTRTRKNYLA (SEQ ID NO: 259) 13B1 parent LC-CDR1 KSSQSLLNSRTRKNYLA(SEQ ID NO: 142) 13B1-NQ LC-CDR1 KSSQSLLQSRTRKNYLA (SEQ ID NO: 257)13B1-NA LC-CDR1 KSSQSLLASRTRKNYLA (SEQ ID NO: 258) 13B1-ST LC-CDR1KSSQSLLNTRTRKNYLA (SEQ ID NO: 259) Consensus for 4D5, LC-CDR1KSSQSLLXXRTRKNYLA 13B1 and variants (SEQ ID NO: 260)Wherein X at position 8 is N, Q or A; and wherein X at position 9 is Sor T 10D12 parent LC-CDR1 KSSQSLLDSDGKTYLN (SEQ ID NO: 153)10D12-DE mutant LC-CDR1 KSSQSLLDSEGKTYLN (SEQ ID NO: 261)10D12-DA mutant LC-CDR1 KSSQSLLDSAGKTYLN (SEQ ID NO: 262)10D12-GA mutant LC-CDR1 KSSQSLLDSDAKTYLN (SEQ ID NO: 263) 35C1 LC-CDR1KSSQSLLDSDGKTYLS (SEQ ID NO: 159) Consensus of 10D12, LC-CDR1KSSQSLLDSXXKTYLX 35C1 and variants (SEQ ID NO: 215)Wherein X at position 10 is D, E or A; Wherein X at position11 is G or A; and wherein X at position 16 is N or S

TABLE 31 Binding of mutagenized candidates of humanized 4D5, 10D12 and13B1 mAbs to human MASP-3 MASP-3 antibody clone Binding to human MASP-3(Fab format) EC₅₀ (pM) h4D5_19-1 parental Fab 102 (VH-19 and VL-1)h4D5-19-1-NQ Fab 732 (VH-19 and VL-1-NQ) h4D5-19-1-NA Fab 122 (VH-19 andVL-1-NA) h4D5-19-1-ST Fab 151 (VH-19 and VL-1-ST) h10D12_45-21 parentalFab 108 (VH-45 and VL-21) h10D12-45-21-DE Fab 326 (VH-45 and VL-21-DE)h10D12-45-21-DA Fab 294 (VH-45 and VL-21-DA) h10D12-45-21-GA Fab 181(VH-45 and VL-21-GA) h13B1_10-1 parental Fab 100 (VH-10 and VL-1)h13B1_10-1-NQ Fab 138 (VH-10 and VL-1-NQ) h13B1_10-1-NA Fab 105 (VH-10and VL-1-NA) h13B1_10-1-ST Fab 120 (VH-10 and VL-1-ST)

TABLE 32MASP-3 Antibody humanized VH Sequences (CDRs and FR regions, Kabat)Antibody HC FR1 HC CDR1 4D5 parent QVQLKQSGPELVKPGASVKLSCKASGYTFT TDDIN(SIN:24) (SEQ ID NO: 55) (SEQ ID NO: 56) h4D5_VH-14QVQLVQSGAEVKKPGASVKVSCKASGYTFT TDDIN (SIN:248) (SEQ ID NO: 264)(SEQ ID NO: 56) h4D5_VH-19 QVQLVQSGAEVKKPGASVKVSCKASGYTFT TDDIN(SIN:249) (SEQ ID NO: 264) (SEQ ID NO: 56) 10D12 parentQIQLVQSGPELKKPGETVKISCKASGYIFT SYGMS (SIN:28) (SEQ ID NO: 71)(SEQ ID NO: 72) h10D12_VH- QIQLVQSGSELKKPGASVKVSCKASGYIFT SYGMS 45(SEQ ID NO: 269) (SEQ ID NO: 72) (SIN:251) hl0D12-VH-QIQLVQSGSELKKPGASVKVSCKASGYIFT SYGMS 49 (SEQ ID NO: 269) (SEQ ID NO: 72)(SIN:252) 13B1 parent QVQLKQSGAELMKPGASVKLSCKATGYTFT GKWIE (SIN:30)(SEQ ID NO: 83) (SEQ ID NO: 84) h13B1_VH-9QVQLVQSGAEVKKPGASVKVSCKASGYTFT GKWIE (SIN:254) (SEQ ID NO: 273)(SEQ ID NO: 84) h13Bl_VH- QVQLVQSGAEVKKPGASVKVSCKASGYTFT GKWIE 10(SEQ ID NO: 273) (SEQ ID NO: 84) (SIN:255) Antibody HC FR2 HC CDR24D5 parent WVKQRPGQGLEWIG WIYPRDDRTKYNDKFKD (SEQ ID NO: 57)(SEQ ID NO: 58) h4D5_VH-14 WVRQAPGQGLEWIG WIYPRDDRTKYNDKFKD(SEQ ID NO: 265) (SEQ ID NO: 58) h4D5_VH-19 WVRQAPGQGLEWIGWIYPRDDRTKYNDKFKD (SEQ ID NO: 265) (SEQ ID NO: 58) 10D12 parentWVRQAPGKGLKWMG WINTYSGVPTYADDFKG (SEQ ID NO: 73) (SEQ ID NO: 74)h10D12_VH- WVRQAPGKGLKWMG WINTYSGVPTYADDFKG 45 (SEQ ID NO: 73)(SEQ ID NO: 74) hl0D12-VH- WVRQAPGKGLKWMG WINTYSGVPTYADDFKG 49(SEQ ID NO: 73) (SEQ ID NO: 74) 13B1 parent WVKQRPGHGLEWIGEILPGTGSTNYNEKFKG (SEQ ID NO: 85) (SEQ ID NO: 86) h13B1_VH-9WVRQAPGQGLEWIG EILPGTGSTNYAQKFQG (SEQ ID NO: 274) (SEQ ID NO: 275)h13B1_VH- WVRQAPGQGLEWIG EILPGTGSTNYNEKFKG 10 (SEQ ID NO: 274)(SEQ ID NO: 86) Antibody HC FR3 HC CDR3 4D5 parentKATLTVDTSSNTAYMDLHSLTSEDSAVYFCSS LEDTY (SEQ ID NO: 59) (SEQ ID NO: 60)h4D5_VH-14 KATLTVDTSSNTAYMELSSLRSEDTAVYYCSS LEDTY (SEQ ID NO: 266)(SEQ ID NO: 60) h4D5_VH-19 RATLTVDTSSNTAYMELSSLRSEDTAVYYCSS LEDTY(SEQ ID NO: 267) (SEQ ID NO: 60) 10D12 parentRFAFSLETSARTPYLQINNLKNEDTATYFCAR GGEAMDY (SEQ ID NO: 75) (SEQ ID NO: 76)h10D12_VH- RFVFSLDTSVRTPYLQISSLKAEDTAVYFCAR GGEAMDY 45 (SEQ ID NO: 270)(SEQ ID NO: 76) hl0D12-VH- RFVFSLDTSVRTPYLQISSLKAEDTATYFCAR GGEAMDY 49(SEQ ID NO: 271) (SEQ ID NO: 76) 13B1 parentKATFTADSSSNTAYMQLSSLTTEDSAMYYCLR SEDV (SEQ ID NO: 87) (SEQ ID NO: 88)h13B1_VH-9 RATFTADSSTSTAYMELSSLRSEDTAVYYCLR SEDV (SEQ ID NO: 276)(SEQ ID NO: 88) h13B1_VH- RATFTADSSTSTAYMELSSLRSEDTAVYYCLR SEDV 10(SEQ ID NO: 276) (SEQ ID NO: 88) Antibody HC FR4 4D5 parent WGQGTLVAVSS(SEQ ID NO: 61) h4D5_VH-14 WGQGTLVTVSS (SEQ ID NO: 268) h4D5_VH-19WGQGTLVTVSS (SEQ ID NO: 268) 10D12 parent WGQGTSVTVSS (SEQ ID NO: 77)h10D12_VH- WGQGTLVTVSS 45 (SEQ ID NO: 272) h10D12-VH- WGQGTLVTVSS 49(SEQ ID NO: 272) 13B1 parent WGTGTTVTVSS (SEQ ID NO: 89) h13B1_VH-9WGQGTLVTVSS (SEQ ID NO: 277) h13B1_VH- WGQGTLVTVSS 10 (SEQ ID NO: 277)

Representative Humanized Light Chain Variable Regions with Variants:

h4D5_VL-1-NA (SEQ ID NO: 278)DIVMTQSPDSLAVSLGERATINCKSSQSLLASRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNL YTFGQGTKVEIKRh10D12_VL-21-GA (SEQ ID NO: 279)DVLMTQTPLSLSVTPGQPASISCKSSQSLLDSDAKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFP WTFGQGTKVEIKRh13B1_VL-1-NA (SEQ ID NO: 280)DIVMTQSPDSLAVSLGERATINCKSSQSLLASRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNI PTFGQGTKVEIKR

TABLE 33 MASP-3 Antibody humanized VL Sequences (CDRs and FR regions,Kabat) [plus variants in LC-CDR1] Antibody LC FR1 LC CDR1 4D5 parentDIVMTQSPSSLAVSAGEKVTMTC KSSQSLLNSRTRKNYLA (SIN:40) (SEQ ID NO: 141)(SEQ ID NO: 142) h4D5_VL-1 DIVMTQSPDSLAVSLGERATINC KSSQSLLNSRTRKNYLA(SIN:250) (SEQ ID NO: 281) (SEQ ID NO: 142) h4D5_VL-1-DIVMTQSPDSLAVSLGERATINC KSSQSLLASRTRKNYLA NA (SEQ ID NO: 281)(SEQ ID NO: 258) (SIN:278) 10D12 DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLN parent (SEQ ID NO: 152) (SEQ ID NO: 153) (SIN:43)h10D12_VL- DVLMTQTPLSLSVTPGQPASISC KSSQSLLDSDGKTYLN 21 (SEQ ID NO: 285)(SEQ ID NO: 153) (SIN:253) h10D12_VL- DVLMTQTPLSLSVTPGQPASISCKSSQSLLDSDAKTYLN 21-GA (SEQ ID NO: 285) (SEQ ID NO: 263) (SIN:279)13B1 parent DIVMTQSPSSLAVSAGEKVTMSC KSSQSLLNSRTRKNYLA (SEQ ID NO: 151)(SEQ ID NO: 142) h13B1_VL-1 DIVMTQSPDSLAVSLGERATINC KSSQSLLNSRTRKNYLA(SEQ ID NO: 281) (SEQ ID NO: 142) h13B1_VL- DIVMTQSPDSLAVSLGERATINCKSSQSLLASRTRKNYLA 1-NA (SEQ ID NO: 281) (SEQ ID NO: 258) Antibody LC FR2LC CDR2 4D5 parent WYQQKPGQSPKLLIY WASTRES (SEQ ID NO: 143)(SEQ ID NO: 144) h4D5_VL-1 WYQQKPGQPPKLLIY WASTRES (SEQ ID NO: 282)(SEQ ID NO: 144) h4D5_VL-1- WYQQKPGQPPKLLIY WASTRES NA (SEQ ID NO: 282)(SEQ ID NO: 144) 10D12 WLLQRPGQSPKRLIY LVSKLDS parent (SEQ ID NO: 154)(SEQ ID NO: 155) h10D12_VL- WLLQRPGQSPKRLIY LVSKLDS 21 (SEQ ID NO: 154)(SEQ ID NO: 155) h10D12_VL- WLLQRPGQSPKRLIY LVSKLDS 21-GA(SEQ ID NO: 154) (SEQ ID NO: 155) 13B1 parent WYQQKPGQSPKLLIY WASTRES(SEQ ID NO: 143) (SEQ ID NO: 144) h13B1_VL-1 WYQQKPGQPPKLLIY WASTRES(SEQ ID NO: 282) (SEQ ID NO: 144) h13B1_VL- WYQQKPGQPPKLLIY WASTRES 1-NA(SEQ ID NO: 282) (SEQ ID NO: 144) Antibody LC FR3 LC CDR3 4D5 parentGVPDRFTGSGSGTDFSLTISSVQAEDLAVYYC KQSYNLYT (SEQ ID NO: 145)(SEQ ID NO: 146) h4D5_VL-1 GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC KQSYNLYT(SEQ ID NO: 283) (SEQ ID NO: 146) h4D5_VL-1-GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC KQSYNLYT NA (SEQ ID NO: 283)(SEQ ID NO: 146) 10D12 GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPWT parent(SEQ ID NO: 156) (SEQ ID NO: 157) h10D12 VL-GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC WQGTHFPWT 21 (SEQ ID NO: 286)(SEQ ID NO: 157) h10D12_VL- GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC WQGTHFPWT21-GA (SEQ ID NO: 286) (SEQ ID NO: 157) 13B1 parentGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC KQSYNIPT (SEQ ID NO: 150)(SEQ ID NO: 161) h13B1_VL-1 GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC KQSYNIPT(SEQ ID NO: 283) (SEQ ID NO: 161) h13Bl_VL-GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC KQSYNIPT 1-NA (SEQ ID NO: 283)(SEQ ID NO: 161) Antibody LC FR4 4D5 parent FGGGTKLEIKR (SEQ ID NO: 147)h4D5_VL-1 FGQGTKVEIKR (SEQ ID NO: 284) h4D5_VL-1- FGQGTKVEIKR NA(SEQ ID NO: 284) 10D12 FGGGTKLEIKR parent (SEQ ID NO: 147) h10D12_VL-FGQGTKVEIKR 21 (SEQ ID NO: 287) h10D12_VL- FGQGTKVEIKR 21-GA(SEQ ID NO: 287) 13B1 parent FGGGTKLEIKR (SEQ ID NO: 147) h13B1_VL-1FGQGTKVEIKR (SEQ ID NO: 284) h13B1_VL- FGQGTKVEIKR 1-NA (SEQ ID NO: 284)

Example 20

Analysis of a representative MASP-3 inhibitory mAb 13B1 in a mouse modelof multiple sclerosis.

Background/Rationale: Experimental autoimmune encephalomyelitis (EAE),an acquired inflammatory and demyelinating autoimmune disorder, is anestablished animal model of multiple sclerosis (MS). Evidence suggestingthat the APC plays a significant role in the development/progression ofEAE was provided by reports that the disease is attenuated in micetreated with a Factor B-neutralizing antibody (Hu et al., Mol. Immunol.54:302, 2013). This Example describes the analysis of a representativehigh affinity MASP-3 inhibitory antibody, 13B1, in the EAE model.

Methods:

EAE Induction:

A kit for inducing EAE, purchased from Hooke Laboratories (Lawrence,Mass.) was used to induce EAE in this study. This kit contained theneuroantigen MOG35-55 in Complete Freund's Adjuvant (CFA) as well aspertussis toxin.

30 wild-type C57B1/6J female mice were used for this study and wereacclimated to the facility for at least one week prior to EAE induction.The mice were approximately 10 weeks of age at the time of induction. Asshown in TABLE 34 below, at the time of induction, each mouse receivedtwo 100 μL subcutaneous (sc) injections of MOG35-55 and oneintraperitoneal (ip) injection of 100 μL (400 ng) pertussis toxin. Asecond injection of pertussis toxin was administered 24 hours after thefirst.

Treatment: The 30 mice were divided into three groups of 10 and treatedwith an irrelevant isotype control mAb 10 mg/kg i.v.); mAb 13B1(anti-MASP-3, 10 mg/kg i.v.) or mAb 1379 (anti-Factor B (Hu et al., Mol.Immunol. 54:302, 2013) 40 mg/kg i.p.). As shown in TABLE 34, dosing withthe isotype control mAb and MASP-3 mAb 13B1 occurred weekly beginning atDay −16 and ending on Day +12. Dosing with mAb 1379 occurred every otherday from Day +3 to Day +11, according to the dosing schedule describedin Hu et al., Mole Immunol 54:302-308, (2013).

TABLE 34 Experimental Methods for EAE Experiment with MASP-3 mAh 13B1Isotype mAh 13B1 Pertussis MOG mAh 1379 Control (anti- Day of Toxinpeptide (anti- mAh MASP-3) Adminis- 400 ng 35-55 Factor B) 10 mg/ 10 mg/tration i.p. 250 μg 40 mg i.p. kg i.v. kg i.v. −16 + + −9 + + −2 + +0 + + +1 + +3 + +5 + + + +7 + +9 + +11 + +12 + +

Scoring: The mice were checked every other day until the emergence ofsymptoms, after which they were checked daily. The first signs ofdisease appeared 7-12 days after immunization, as expected. The micewere scored according to the scale shown below in TABLE 35.

TABLE 35 EAE Model Scoring Criteria Score Clinical Observations 0.0 Noobvious changes in motor functions of the mouse in comparison tonon-immunized mice. When picked up by the base of the tail, the tail hastension and is erect. Hind legs are usually spread apart. When the mouseis walking, there is no gait or head tilting. 0.5 Tip of tail is limp.When the mouse is picked up by the base of the tail, the tail hastension except for the tip. Muscle straining is felt in the tail, whilethe tail continues to move. 1.0 Limp tail. When mouse is picked up bythe base of the tail, instead of being erect, the whole tail drapes overfinger. Hind legs are usually spread apart. No signs of tail movementare observed. 1.5 Limp tail and hind leg inhibition. When picked up bythe base of the tail, the whole tail drapes over finger. When the mouseis dropped on a wire rack, at least one hind leg falls throughconsistently. Walking is very slightly wobbly. 2.0 Limp tail andweakness of hind legs. When picked up by the base of the tail, the legsare not spread apart, but held close together. When the mouse isobserved walking, it has a clearly apparent wobbly walk. One foot mayhave toes dragging, but the other leg has no apparent inhibitions ofmovement; OR, Mouse appears to be at score 0.0, but there are obvioussigns of head tilting when the walk is observed. The balance is poor.2.5 Limp tail and dragging of hind legs. Both hind legs have somemovement, but both are dragging at the feet (mouse trips on hindfeet). - OR- No movement in one leg/completely dragging one leg, butmovement in the other leg.- OR - EAE severity appears mild when pickedup (as score 0.0-1.5), but there is a strong head tilt that causes themouse to occasionally fall over. 3.0 Limp tail and complete paralysis ofhind legs (most common). - OR - Limp tail and almost complete paralysisof hind legs. One or both hind legs are able to paddle, but neither hindleg is able to move forward of the hind hip. - OR - Limp tail withparalysis of one front and one hind leg. - OR - ALL of: Severe headtilting, walking only along the edges of the cage, pushing against thecage wall, spinning when picked up by base of tail. 3.5 Limp tail andcomplete paralysis of hind legs. In addition to: Mouse is moving aroundthe cage, but when placed on its side, is unable to right itself. Hindlegs are together on one side of body. - OR - Mouse is moving around thecage, but the hind quarters are flat like a pancake, giving theappearance of a hump in the front quarters of the mouse 4.0 Limp tail,complete hind leg and partial front leg paralysis. Mouse is minimallymoving around the cage but appears alert and feeding. Often euthanasiais recommended after the mouse scores 4.0 for 2 days. However, withdaily s.c. fluids some mice can recover to 3.5 or 3.0. When the mouse iseuthanized because of severe paralysis, a score of 5.0 is entered forthat mouse for the rest of the experiment. 4.5 Complete hind and partialfront leg paralysis, no movement around the cage. Mouse is not alert.Mouse has minimal movement in the front legs. The mouse barely respondsto contact. Euthanasia is recommended. When the mouse is euthanizedbecause of severe paralysis, a score of 5.0 is entered for that mousefor the rest of the experiment. 5.0 Mouse is spontaneously rolling inthe cage.

Results:

FIG. 68 graphically illustrates the results of the EAE model in micetreated with either MASP-3 inhibitory mAb 13B1 (10 mg/kg), Factor B mAb1379 (40 mg/kg) or isotype control mAb (10 mg/kg), wherein downwardpointing arrows indicate dosing of anti-Factor B antibody and upwardpointing arrows indicates the last dose of mAb 13B1. As shown in FIG.68, mice treated with MASP-3 inhibitory mAb 13B1 and Factor B mAb 1379exhibited an improvement in clinical symptoms scored according to theparameters shown in TABLE 35, as compared to isotype control.

In accordance with the foregoing, MASP-3 inhibitory antibodies, such asthe high affinity MASP-3 inhibitory antibodies disclosed herein, areexpected to be beneficial (neuroprotective or neuroregenerative) in thetreatment and/or rehabilitation of a subject suffering from multiplesclerosis, Balo concentric sclerosis, neuromyelitis optica, Marburgmultiple sclerosis, Schilder's disease, Tumefactive multiple sclerosisand acute disseminated encephalomyelitis (ADM).

Example 21

Pharmacodynamic Study with Representative High Affinity MASP-3 mAbs inCynomolgus Monkeys.

Background/Rationale: As was demonstrated in rodent studies (FIG. 44), ahigh affinity MASP-3 inhibitory antibody was capable of inhibitingsteady-state (resting) pro-factor D maturation in vivo. This Exampledescribes a study that was carried out in cynomolgus monkeys todetermine if representative high affinity MASP-3 inhibitory mAbs arecapable of inhibiting APC activity in a non-human primate.

Methods: To confirm that MASP-3 functions in the APC in a non-humanprimate, and that the high affinity MASP-3 antibodies are capable ofinhibiting the APC in a non-human primate, 9 cynomolgus monkeys (3animals per mAb condition) were given a single 5 mg/kg intravenous dosewith one of three representative high affinity MASP-3 inhibitoryantibodies: h4D5X, h10D12X, or h13B1X. (“h” refers to humanized, “X”refers to the IgG4 constant hinge region (SEQ ID NO:312) containing thestabilizing S228P amino acid substitution and a mutation human IgG4constant region with S228P mutation and also a mutation that promotesFcRn interations at low pH). Plasma (EDTA) and serum samples werecollected at regular intervals over a period of three weeks or longer.

Two assays were employed to measure APC activity in the sera fromtreated monkeys. The first assay assessed levels of complement factor Bbdeposited on zymosan beads added to diluted serum. The second assaymeasured the fluid phase products of the zymosan-activated APC,complement factors Ba and Bb, as well as C3a.

Flow cytometry using the factor Bb antibody A252 (Quidel) was used todetect factor Bb deposited on zymosan. As a means for determining thebackground signal in the assay following complete inhibition of the APC,an aliquot of serum (5% final, diluted in GVB +Mg/EGTA) prepared fromMASP-3 mAb-treated cynomolgus monkeys was spiked with 300 nM of aninhibitory Factor D antibody. To determine the degree of APC inhibitionby the MASP-3 mAb delivered intravenously to the monkey, another aliquotof diluted serum was spiked with 300 nM of a neutral isotype controlantibody (that has no APC inhibitory activity) before testing factor Bbdeposition on zymosan. The spiked antibody-serum mixtures were incubatedfor 30 minutes on ice prior to the addition of zymosan (0.1 mg/mLfinal). The mixtures were incubated at 37° C. for 65 minutes, and theAPC activity was measured by the flow cytometric detection of complementfactor Bb (Quidel antibody A252) on the surface of the zymosanparticles.

For determining generation of the fluid phase markers Ba, Bb, and C3a,the APC was induced in ex vivo assays by incubating zymosan (1 mg/mLfinal) in serum (5% final, diluted in GVB+Mg/EGTA) prepared fromanti-MASP-3 mAb-treated cynomolgus monkeys. The mixtures were incubatedat 37° C. for 40 minutes, and the APC activity was measured byELISA-based detection of the complement end-points. Ba, Bb, and C3a weredetected in the reaction supernatants using commercially available ELISAkits (Quidel). Absorbance values of all tests were normalized by settingpre-treatment values as 100% activity, and a pre-treatment sampleincubated, but not exposed to zymosan, to 0%.

In order to relate the degree of APC inhibition to the antibody totarget ratio in MASP-3 mAb-treated monkeys, serum MASP-3 and inhibitoryMASP-3 mAb levels were quantitated. Serum MASP-3 was measured by asandwich ELISA assay. The MASP-3 protein was captured on a plate withαM3-259 (described in Example 16). Serum samples (diluted 1:40) werefirst incubated with unlabeled (non-biotinylated) MASP-3 mAb,corresponding to the treatment mAb, at 37° C. for 1 hour, then furtherdiluted 1:250 (final 1:10,000) and added to the plate and incubated at37° C. for another hour. The plate was washed and a biotinylated versionof mAb 10D12 was used as a detection antibody. The large dilution ofserum prior to the detection steps was used to uncouple target andtreatment mAb, and to prevent competition between the treatment antibodyand the detection antibody. After the plate was washed multiple times,streptavidin-HRP was used for the final detection step. Absorbancevalues were collected at A450 with a plate reader. MASP-3 serumconcentrations were extrapolated from a standard curve created byassaying recombinant, full-length cyno MASP-3 protein. The amount ofanti-MASP-3 antibody present in the serum was detected using the HumanTherapeutic IgG4 ELISA Kit (Cayman Chemicals), following themanufacturer's instructions.

Western blot analysis was used to analyze the level of pro-Factor D andFactor D in serum from a cynomolgus monkey over time (hours) aftertreatment with a single 5 mg/kg intravenous dose of mAb h13B1X. Brieflydescribed, the Western blot analysis was carried out by mixing 20 μL ofcynomolgus plasma obtained at the different timepoints prior totreatment (−120 hr, −24 hr) and after treatment (72 hr, 168 hr, 336 hr,504 hr, 672 hr and 840 hr) with PBS and 11.2 μL of anti-CFD antibody(0.5 μg/μL) in a total volume of 4004, at 4° C. for 1 hour. 12 μL ofProtein A/G Plus Agarose (Santa Cruz Biotech) was added and the mixturewas incubated overnight at 4° C. Immunoprecipitates were collected bycentriguation at 1000×g for 5 minutes at 4° C. The pellets were washedfive times with PBS. After the final wash, the pellets were resuspendedin 30 μL of lx Glycoprotein Denaturing Buffer and the glycoprotein wasdenatured by heating the reaction at 100° C. for 10 minutes. 10X G2reaction buffer, 10% NP-40 and 2.5 μL Peptide-N-Glycosidase (New EnglandBiolabs, P0704L) was added into each tube and the reaction was incubatedat 37° C. for 2 hours. The agarose beads were pelleted by centrifugationat 1000×g for 5 minutes and 20 μL supernatant was collected into newtubes. The captured and deglycosylated proteins were resolved withSDS-PAGE (NuPAGE 12% Bis-Tris Mini Gel) and the gels were electroblottedfor Western blot analysis with a biotinylated anti-CFD (R&D SystemsBAF1824) and Pierce™ High Sensitivity Streptavidin-HRP (Thermo FischerScientific 21130).

Results:

FIG. 69 graphically illustrates APC activityin serum samples obtainedfrom a group of three cynomolgus monkeys over time after a singletreatment at time=0 with high affinity MASP-3 mAb h13B1X. The figureshows the average MFI in a flow cytometric assay detecting complementfactor Bb on the surface of zymosan particles in 5% serum spiked witheither the APC-inhibiting fact D mAb or the neutral isotype control mAb.As shown in FIG. 69, the animals demonstrate diminished APC activity asearly as 4 hrs. If MASP-3 antibody treatment blocks the APC aseffectively as Factor D inhibition, the two spiked antibody conditionswill demonstrate identical levels of inhibition of Bb deposition inpost-dose samples, but not in the pre-dose (or time=0; FIG. 69)condition. As shown in FIG. 69, by 72 hrs post-treatment, the APCactivity is decreased to approximately that achieved by adding theFactor D mAb to the serum samples. Nearly complete inhibition due toh13B1X treatment, as experimentally determined by comparison with thespiked Factor D antibody, persists until 336 hrs (14 days) post-dose.Thus, these results demonstrate that treatment with a high affinityMASP-3 inhibitory mAb provides a complete, sustained inhibition of theAPC in a non-human primate.

FIG. 70 graphically illustrates APC activity, as determined by Bbdeposition on zymosan, in serum samples obtained from groups ofcynomolgus monkeys (3 animals per group) treated with a single 5 mg/kgintravenous dose of high affinity MASP-3 inhibitory mAbs h4D5X, h10D12Xor h13B1X. Bb deposition data was collected as described above. APCactivity for the treatment timepoints was normalized by settingpre-treatment MFIs of samples spiked with the non-inhibitory, isotypecontrol antibody as 100% activity, and a pre-treatment sample incubatedwith 50 mM EDTA (to inhibit all complement activity) to 0%. The h13BXtreatment data used for FIG. 70 are also reflected in FIG. 69. As shownin FIG. 70, treatment with all three high affinity MASP-3 inhibitoryantibodies resulted in greater than 95% inhibition of the APC. Theh4D5X-, h10D12X-, and h13B1X-treated animals maintained at least 90%inhibition of the APC for 6.7, 11.7, and 16 days, respectively. Thus,these results demonstrate that treatment with these represensative highaffinity MASP-3 inhibitory mAbs provides sustained inhibition of the APCin a nonhuman primate with a single 5 mg/kg dose.

FIG. 71A-C graphically illustrates additional measures of APC activity.Fluid-phase Ba (FIG. 71A), Bb (FIG. 71B) and C3a (FIG. 71C) weremeasured in zymosan-treated, diluted serum samples obtained from groupsof cynomolgus monkeys (3 animals per group) over time after treatmentwith a single 5 mg/kg intravenous dose of h4D5X, h10D12X, and h13B1X asdescribed above.

As shown in FIG. 71A-C, single administrations of all three highaffinity MASP-3 inhibitory antibodies resulted in inhibition of the APC,as defined by three different fluid-phase endpoints. These data areconsistent with level of APC inhibition demonstrated in the Bbdeposition study of FIG. 70, and further illustrate the efficacy ofthese mAbs to inhibit the pathway for multiple weeks.

FIG. 72A-C graphically illustrates the relationship of APC activity, asdetermined by fluid-phase Ba production, relative to the molar ratio ofmonomeric MASP-3 and MASP-3 mAb antibody detected in serum from monkeystreated with either h4D5X (FIG. 72A), h10D12X (FIG. 72B) or h13B1X (FIG.72C). Each panel in FIG. 72A-C represents the data from one monkey. Themonkey subjects used and serum (or plasma) obtained in this study arethe same as those described above (FIGS. 69, 70, and 71).

FIGS. 72A-C graphically illustrates the molar ratio of target (MASP-3)to the high affinity MASP-3 inhibitory antibodies h4D5X (FIG. 72A),h10D12X (FIG. 72B) and h13B1X (FIG. 72C) at the timepoints of completeAPC inhibition, as measured by fluid-phase Ba. For reference purposes,the molar ratio of 1:1 target to antibody is shown as a dotted line ineach graph. As shown in FIGS. 72A-C, target (MASP-3) to the highaffinity MASP-3 inhibitory mAbs h4D5X, h10D12X and h13B1X at a molarratio in the range of about 2:1 to about 2.5:1 (target to antibody) aresufficient to completely inhibit the APC. These data demonstrate thatthese three representative MASP-3 inhibitory mAbs are potent,high-affinity MASP-3 inhibitory antibodies that are capable atinhibiting the APC when present at molar concentrations less than theconcentration of target. These levels of potency strongly indicate thatthe mAbs have the potential to be used clinically to treat diseases orindications caused by the APC.

FIG. 73 shows a Western blot analyzing the level of pro-Factor D andFactor D in serum from a cynomolgus monkey over time (hours) prior toand after treatment with a single 5 mg/kg intravenous dose of mAbh13B1X. As shown in FIG. 73, Factor D is present in plasma as pro-FactorD for at least 336 hours (14 days) following a single dose of mAbh13B1X.

Summary of Results

As described in Example 11, a single dose administration of a highaffinity MASP-3 inhibitory antibody, mAb 13B1, to mice led tonear-complete ablation of systemic alternative pathway complementactivity for at least 14 days. As further described in Example 12, in astudy conducted in a well-established animal model associated with PNHit was demonstrated that mAb 13B1 significantly improved the survival ofPNH-like red blood cells and protected PNH-like red blood cellssignificantly better than did C5 inhibition. As described in Example 13,it was further demonstrated that mAb 13B1 reduced the incidence andseverity of disease in a mouse model of arthritis. The results in thisexample demonstrate that representative high affinity MASP-3 inhibitorymAbs 13B1, 10D12 and 4D5 are highly effective at blocking thealternative pathway in primates. Single dose administration of mAb 13B1,10D12 or 4D5 to cynomolgus monkeys resulted in sustained ablation ofsystemic alternative pathway activity lasting for approximately 16 days.The extent of alternative pathway ablation in cynomolgus monkeys treatedwith high affinity MASP-3 inhibitory antibodies was comparable to thatachieved by factor D blockade in vitro, indicating complete blockade offactor D conversion by the MASP-3 inhibitory antibodies. Therefore, highaffinity MASP-3 inhibitory mAbs have therapeutic utility in thetreatment of patients suffering from diseases related to alternativepathway hyperactivity, such as, for example, paroxysmal nocturnalhemoglobinuria (PNH), age-related macular degeneration (AMD, includingwet and dry AMD), ischemia-reperfusion injury, arthritis, disseminatedintravascular coagulation, thrombotic microangiopathy (includinghemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome(aHUS),thrombotic thrombocytopenic purpura (TTP) ortransplant-associated TMA), asthma, dense deposit disease, pauci-immunenecrotizing crescentic glomerulonephritis, traumatic brain injury,aspiration pneumonia, endophthalmitis, neuromyelitis optica, Behcet'sdisease, multiple sclerosis, Guillain Barre Syndrome, Alzheimer'sdisease, Amylotrophic lateral sclerosis (ALS), lupus nephritis, systemiclupus erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronicobstructive pulmonary disease (COPD), C3 glomerulopathy, transplantrejection, Graft-versus-host disease (GVHD), hemodialysis, sepsis,Systemic inflammatory response syndrome (SIRS), Acute RespiratoryDistress Syndrome (ARDS), ANCA vasculitis, Anti-phospholipid syndrome,Atherosclerosis, IgA Nephropathy and Myasthenia Gravis.

VII. OTHER EMBODIMENTS

All publications, patent applications, and patents mentioned in thisspecification are herein incorporated by reference.

Various modifications and variations of the described methods,compositions, and compounds, of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific desired embodiments, it should be understood that the inventionas claimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention that are obvious to those skilled in the fields ofmedicine, immunology, pharmacology, oncology, or related fields areintended to be within the scope of the invention.

In accordance with the foregoing, the invention features the followingembodiments.

High Affinity MASP-3 Inhibitory Antibodies that Bind One or MoreEpitopes within the SP Domain

1A. An isolated monoclonal antibody or antigen-binding fragment thereofthat specifically binds to the serine protease domain of human MASP-3(amino acid residues 450 to 728 of SEQ ID NO:2) with high affinity(having a K_(D) of less than 500 pM), wherein the antibody orantigen-binding fragment thereof inhibits alternative pathway complementactivation.

2A. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the antibody or antigen-binding fragment ischaracterized by at least one or more of the following properties:

-   -   (a) inhibits pro-Factor D maturation;    -   (b) does not bind to human MASP-1 (SEQ ID NO:8);    -   (c) inhibits the alternative pathway at a molar ratio of from        about 1:1 to about 2.5:1 (MASP-3 target to mAb) in a mammalian        subject;    -   (d) does not inhibit the classical pathway.    -   (e) inhibition of hemolysis and/or opsonization;    -   (f) inhibition of MASP-3 serine protease substrate-specific        cleavage;    -   (g) a reduction of hemolysis or the reduction of C3 cleavage and        C3b surface deposition;    -   (h) a reduction of Factor B and Bb deposition on an activating        surface;    -   (i) a reduction of resting levels (in circulation, and without        the experimental addition of an activating surface) of active        Factor D relative to pro-Factor D;    -   (j) a reduction of levels of active Factor D relative to        pro-Factor D in response to an activating surface;    -   (k) a reduction of the production of resting and/or        surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a and/or    -   (l) a reduction in factor P deposition.

3A. The isolated antibody or antigen-binding fragment thereof ofparagraph 1 or 2, wherein said antibody or antigen-binding fragmentthereof specifically binds to an epitope located within the serineprotease domain of human MASP-3, wherein said epitope is located withinat least one or more of: VLRSQRRDTTVI (SEQ ID NO:9), TAAHVLRSQRRDTTV(SEQ ID NO:10), DFNIQNYNHDIALVQ (SEQ ID NO:11), PHAECKTSYESRS (SEQ IDNO:12), GNYSVTENMFC (SEQ ID NO:13), VSNYVDWVWE (SEQ ID NO:14) and/orVLRSQRRDTTV (SEQ ID NO:15). [Group I]

4A. The antibody or antigen-binding fragment thereof of paragraph 3,wherein said antibody or antigen-binding fragment binds to an epitopewithin SEQ ID NO:15. [includes all group I abs]

5A. The antibody or antigen-binding fragment of paragraph 3, whereinsaid antibody or antigen-binding fragment binds to an epitope within SEQID NO:9. [10D12]

6A. The antibody or antigen-binding fragment of paragraph 3, whereinsaid antibody or antigen-binding fragment binds to an epitope within SEQID NO:10. [13B1]

7A. The antibody or antigen-binding fragment of paragraph 6, whereinsaid antibody or antigen binding fragment also binds to an epitopewithin SEQ ID NO:12. [13B1]

8A. The antibody or antigen-binidng fragment of paragraph 3, whereinsaid antibody or antigen-binding fragment also binds to an epitopewithin SEQ ID NO:10 and/or SEQ ID NO:12. [13B1]

9A. The antibody or antigen-binding fragment of paragraph 3, whereinsaid antibody or antigen binding fragment binds to an epitope within SEQID NO:9. [1F3, 4B6, 4D5, 1A10]

10A. The antibody or antigen-binding fragment of paragraph 7, whereinsaid antibody or antigen binding fragment also binds to an epitopewithin at least one of SEQ ID NO:11, SEQ ID NO: 13 and/or SEQ ID NO:14.[1F3, 4B6, 4D5, 1A10]

11A. The antibody or antigen-binding fragment of paragraph 7, whereinthe antibody or antigen-binding fragment also binds to an epitope withinat least one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and/or SEQ IDNO:14. [1F3, 4B6, 4D5, 1A10]

12A. The antibody or antigen-binding fragment of paragraph 1 or 2,wherein said antibody binds to an epitope within at least one of:ECGQPSRSLPSLV (SEQ ID NO:16), RNAEPGLFPWQ (SEQ ID NO:17);KWFGSGALLSASWIL(SEQ ID NO:18); EHVTVYLGLH (SEQ ID NO:19); PVPLGPHVMP(SEQ ID NO:20); APHMLGL (SEQ ID NO:21); SDVLQYVKLP (SEQ ID NO:22);and/or AFVIFDDLSQRW (SEQ ID NO:23). [group II and III]

13A. The antibody or antigen-binding fragment of paragraph 12, whereinsaid antibody or antigen-binding fragment binds to an epitope within SEQID NO:17. [all group II and III abs]

14A. The antibody or antigen-binding fragment of paragraph 13, whereinsaid antibody or antigen binding fragment also binds to an epitopewithin EHVTVYLGLH (SEQ ID NO:19) and/or AFVIFDDLSQRW (SEQ ID NO:23).[1G4, 1E7, 2D7 15D9]

15A. The antibody or antigen-binding fragment of paragraph 14, whereinsaid antibody or antigen binding fragment also binds to an epitopewithin SEQ ID NO:23. [1G4, 1E7, 2D7, 15D9, 2F 5]

16A. The antibody or antigen-binding fragment of paragraph 14, whereinsaid antibody or antigen binding fragment also binds to an epitopewithin SEQ ID NO:19 and/or SEQ ID NO:23. [Ig4, 1E7, 2D7]

17A. The antibody or antigen-binding fragment of paragraph 14, whereinsaid antibody or antigen-binding fragment also binds to an epitopewithin SEQ ID NO:18, SEQ ID NO:20 and/or SEQ ID NO:23. [15D9, 2F5]

18A. The antibody or antigen-binding fragment of paragraph 14, whereinsaid antibody or antigen-binding fragment also binds to an epitopewithin at least one of SEQ ID NO:18, SEQ ID NO:20 and/or SEQ ID NO:23[15D9, 2F5].

19A. The antibody or antigen-binding fragment of paragraph 14, whereinsaid antibody or antigen-binding fragment also binds to an epitopewithin at least one of SEQ ID NO:16, SEQ ID NO: 21 and/or SEQ ID NO:22.[1B11]

20A. The antibody or antigen-binding fragment of paragraph 14, whereinsaid antibody or antigen-binding fragment also binds to an epitopewithin at least one of SEQ ID NO:16, SEQ ID NO: 21 and/or SEQ ID NO[1B11].

21A. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-20, wherein the antibody or antigen-binding fragment isselected from the group consisting of a human antibody, a humanizedantibody, a chimeric antibody, a murine antibody, and an antigen-bindingfragment of any of the foregoing.

22A. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-21, wherein said antibody or antigen binding fragmentthereof is selected from the group consisting of a single chainantibody, an ScFv, a Fab fragment, an Fab′ fragment, an F(ab′)2fragment, a univalent antibody lacking a hinge region and a wholeantibody.

23A. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-22, further comprising an immunoglobulin constant region.

24A. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-23, wherein the antibody or antigen-binding fragment ishumanized.

25A. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-24 wherein said antibody binds to the serine proteasedomain of human MASP-3 with an affinity of less than 500 pM.

26A. The antibody or antigen-binding fragment thereof of any ofparagraphs 1-25, wherein said antibody inhibits alternative pathwayactivation in mammalian blood.

27A. A composition comprising the antibody or antigen-binding fragmentof any of paragraphs 1A-26A and a pharmaceutically acceptable excipient.

A. Group IA High Affinity MASP-3 Inhibitory Antibodies that Bind One orMore Epitopes within the SP Domain (4D5, 4B6, 1A10 plus 4D5 Variants)

1B. An isolated antibody, or antigen-binding fragment thereof, thatbinds to MASP-3 comprising:

-   -   (a) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:209 (XXDIN, wherein X at position 1 is S or T and        wherein X at position 2 is N or D); a HC-CDR2 set forth as SEQ        ID NO:210 (WIYPRDXXXKYNXXFXD, wherein X at position 7 is G or D;        X at position 8 is S, T or R; X at position 9 is I or T; X at        position 13 is E or D; X at position 14 is K or E; and X at        position 16 is T or K); and a HC-CDR3 set forth as SEQ ID NO:211        (XEDXY, wherein X at position 1 is L or V, and wherein X at        position 4 is T or S); and    -   (b) a light chain variable region comprising a LC-CDR1 set forth        as SEQ ID NO:212 (KSSQSLLXXRTRKNYLX, wherein X at position 8 is        N, I, Q or A; wherein X at position 9 is S or T; and wherein X        at position 17 is A or S); a LC-CDR2 set forth as SEQ ID NO:144        (WASTRES) and a LC-CDR3 set forth as SEQ ID NO:146 (KQSYNLYT).

2B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR1 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:56 (TDDIN). [4D5 and variants]

3B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR1 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:62 (SNDIN). [1F3, 4B6 and 1A10]

4B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR2 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:58 (WIYPRDDRTKYNDKFK_(D)) [4D5 andvariants].

5B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR2 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:63 (WIYPRDGSIKYNEKFTD). [1F3]

6B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR2 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:67 (WIYPRDGTTKYNEEFTD). [4B6]

7B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR2 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:69 (WIYPRDGTTKYNEKFTD). [1A10]

8B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR3 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:60 (LEDTY)[4D5 and variants]

9B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR3 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:65 (VEDSY). [1F3, 4B6 and 1A10]

10B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the LC-CDR1 of the light chain variable regionaccording to (b) comprises SEQ ID NO:142 (KSSQSLLNSRTRKNYLA); SEQ IDNO:257 (KSSQSLLRTRKNYLA), SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ IDNO:259 (KSSQSLLNTRTRKNYLA). [4D5 and variants]

11B. The isolated antibody or antigen-binding fragment thereof ofparagraph 10, wherein the LC-CDR1 of the light chain variable regionaccording to (b) comprises SEQ ID NO:258 (KSSQSLLASRTRKNYLA). [4D5 NAmutant]

12B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the LC-CDR1 of the light chain variable regionaccording to (b) comprises SEQ ID NO:149 (KSSQSLLISRTRKNYLS). [1F3 and4B6]

13B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR1 comprises SEQ ID NO:56, the HC-CDR2comprises SEQ ID NO:58, the HC-CDR3 comprises SEQ ID NO:60 and whereinthe LC-CDR1 comprises SEQ ID NO:142, SEQ ID NO:257, SEQ ID NO:258 or SEQID NO:259; wherein the LC-CDR2 comprises SEQ ID NO:144 and wherein theLC-CDR3 comprises SEQ ID NO:146. [all 6 CDRs of 4D5 with variants atLC-CDR1].

14B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR1 comprises SEQ ID NO:62, the HC-CDR2comprises SEQ ID NO:63, SEQ ID NO:67 or SEQ ID NO:69, the HC-CDR3comprises SEQ ID NO:65 and wherein the LC-CDR1 comprises SEQ ID NO:149,the LC-CDR2 comprises SEQ ID NO:144 and the LC-CDR3 comprises SEQ IDNO:146. [all 6 CDRS of 1F3, 4B6 and 1A10]

15B. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-14, wherein the antibody or antigen-binding fragment isselected from the group consisting of a human antibody, a humanizedantibody, a chimeric antibody, a murine antibody, and an antigen-bindingfragment of any of the foregoing.

16B. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-15, wherein said antibody or antigen binding fragmentthereof is selected from the group consisting of a single chainantibody, an ScFv, a Fab fragment, an Fab′ fragment, an F(ab′)2fragment, a univalent antibody lacking a hinge region and a wholeantibody.

17B. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-16, further comprising an immunoglobulin constant region.

18B. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-17, wherein the antibody or antigen-binding fragment ishumanized.

19B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to SEQ ID NO:24, SEQ ID NO:248 or SEQ ID NO:249 and alight chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:40, SEQ ID NO:250 or SEQ ID NO:278 [4D5 parental,humanized and modified versions].

20B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to SEQ ID NO:25 and a light chain comprising at least80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:41 [1F3].

21B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to SEQ ID NO:26 and a light chain comprising at least80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:42 [4B6].

22B. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to SEQ ID NO:27 and a light chain comprising at least80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:42 [1A10].

23B. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-22 wherein said antibody binds to human MASP-3 with anaffinity of less than 500 pM.

24B. The antibody or antigen-binding fragment thereof of any ofparagraphs 1-23, wherein said antibody inhibits alternative pathwayactivation in mammalian blood.

25B. A composition comprising the antibody or antigen-binding fragmentof any of paragraphs 1B-24B and a pharmaceutically acceptable excipient.

B. Group IB High Affinity MASP-3 Inhibitory Antibodies that Bind One orMore Epitopes within the SP Domain (10D12, 35C1 and 10D12 Variants)

1C. An isolated antibody, or antigen-binding fragment thereof, thatbinds to MASP-3 comprising:

-   -   (a) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:213 (SYGXX, wherein X at position 4 is M or I and        wherein X at position 5 is S or T); a HC-CDR2 set forth as SEQ        ID NO:74; and a HC-CDR3 set forth as SEQ ID NO:214 (GGXAXDY,        wherein X at position 3 is E or D and wherein X at position 5 is        M or L); and    -   (b) a light chain variable region comprising a LC-CDR1 set forth        as SEQ ID NO:215 (KSSQSLLDSXXKTYLX , wherein X at position 10 is        D, E or A; wherein X at position 11 is G or A; and wherein X at        position 16 is N or S); a LC-CDR2 set forth as SEQ ID NO:155;        and a LC-CDR3 set forth as SEQ ID NO:216 (WQGTHFPXT, wherein X        at position 8 is W or Y).

2C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR1 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:72 (SYGMS). [10D12 and variants]

3C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR1 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:79 (SYGIT). [35C1]

4C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR3 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:76 (GGEAMDY). [10D12 and variants].

5C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR3 of the heavy chain variable regionaccording to (a) comprises SEQ ID NO:82 GGDALDY). [35C1]

6C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the LC-CDR1 of the light chain variable regionaccording to (b) comprises SEQ ID NO:153 (KSSQSLLDSDGKTYLN); SEQ IDNO:261 (KSSQSLLDSEGKTYLN), SEQ ID NO:262 (KSSQSLLDSAGKTYLN) or SEQ IDNO:263 (KSSQSLLDSDAKTYLN). [10D12 and variants]

7C. The isolated antibody or antigen-binding fragment thereof ofparagraph 6, wherein the LC-CDR1 of the light chain variable regioncomprises SEQ ID NO:263 (KSSQSLLDSDAKTYLN). [10D12 GA variant]

8C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the LC-CDR1 of the light chain variable regioncomprises SEQ ID NO:152. [35C1]

9C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the LC-CDR3 of the light chain variable regionaccording to (b) comprises SEQ ID NO:159 (KSSQSLLDSDGKTYLS). [10D12]

10C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the LC-CDR3 of the light chain variable regionaccording to (b) comprises SEQ ID NO:160 (WQGTHFPYT). [35C1]

11C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR1 comprises SEQ ID NO:72, the HC-CDR2comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:76, the LC-CDR1comprises SEQ ID NO:153, SEQ ID NO:261, SEQ ID NO:262 or SEQ ID NO:263;the LC-CDR2 comprises SEQ ID NO:155 and the LC-CDR3 comprises SEQ IDNO:157. [all 6 CDRs of 10D12 with variants at LC-CDR1]

12C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the HC-CDR1 comprises SEQ ID NO:79, the HC-CDR2comprises SEQ ID NO:74, the HC-CDR3 comprises SEQ ID NO:82, the LC-CDR1comprises SEQ ID NO:159, the LC-CDR2 comprises SEQ ID NO:155 and theLC-CDR3 comprises SEQ ID NO:160. [all 6 CDRs of 35C1]

13C. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-12, wherein the antibody or antigen-binding fragment isselected from the group consisting of a human antibody, a humanizedantibody, a chimeric antibody, a murine antibody, and an antigen-bindingfragment of any of the foregoing.

14C. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-13, wherein said antibody or antigen binding fragmentthereof is selected from the group consisting of a single chainantibody, an ScFv, a Fab fragment, an Fab′ fragment, an F(ab′)2fragment, a univalent antibody lacking a hinge region and a wholeantibody.

15C. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-14, further comprising an immunoglobulin constant region.

16C. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-15, wherein the antibody or antigen-binding fragment ishumanized.

17C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to SEQ ID NO:28, SEQ ID NO:251 or SEQ ID NO:252 and alight chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:43, SEQ ID NO:253 or SEQ ID NO:279 [10D12parental, humanized and variants].

18C. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to SEQ ID NO:29 and a light chain comprising at least80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:44 [35C1].

19C. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-18 wherein said antibody binds to human MASP-3 with anaffinity of less than 500 pM.

20C. The antibody or antigen-binding fragment thereof of any ofparagraphs 1-19, wherein said antibody inhibits alternative pathwayactivation in mammalian blood.

21C. A composition comprising the antibody or antigen-binding fragmentof any of paragraphs 1C-20C and a pharmaceutically acceptable excipient.

C. Group IC High Affinity MASP-3 Inhibitory Antibodies that Bind One orMore Epitopes within the SP Domain (13B1 and Variants)

1D. An isolated antibody, or antigen-binding fragment thereof, thatbinds to MASP-3 comprising:

-   -   (a) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:84 (GKWIE); a HC-CDR2 set forth as SEQ ID NO:86        (EILPGTGSTNYNEKFKG) or SEQ ID NO:275 (EILPGTGSTNYAQKFQG); and a        HC-CDR3 set forth as SEQ ID NO:88 (SEDV); and    -   (b) a light chain variable region comprising a LC-CDR1 set forth        as SEQ ID NO:142 (KSSQSLLNSRTRKNYLA), SEQ ID NO:257        (KSSQSLLQSRTRKNYLA); SEQ ID NO:258 (KSSQSLLASRTRKNYLA); or SEQ        ID NO:259 (KSSQSLLNTRTRKNYLA), a LC-CDR2 set forth as SEQ ID        NO:144 (WASTRES); and a LC-CDR3 set forth as SEQ ID NO:161        (KQSYNIPT). [all 6 CDRs of 13B1 and variants in LC-CDR1]

2D. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the LC-CDR1 comprises SEQ ID NO:258. [13B1 LC-CDR1NA variant]

3D. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-2, wherein the antibody or antigen-binding fragment isselected from the group consisting of a human antibody, a humanizedantibody, a chimeric antibody, a murine antibody, and an antigen-bindingfragment of any of the foregoing.

4D. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-3, wherein said antibody or antigen binding fragmentthereof is selected from the group consisting of a single chainantibody, an ScFv, a Fab fragment, an Fab′ fragment, an F(ab′)2fragment, a univalent antibody lacking a hinge region and a wholeantibody.

5D. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-4, further comprising an immunoglobulin constant region.

6D. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-5, wherein the antibody or antigen-binding fragment ishumanized.

7D. The isolated antibody or antigen-binding fragment of paragraph 1,wherein the antibody or antigen-binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:30, SEQ ID NO:254 or SEQ ID NO:255 and a lightchain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identicalto SEQ ID NO:45, SEQ ID NO:256 or SEQ ID NO:280 [13B1 parental,humanized and variants].

8D. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-7 wherein said antibody binds to human MASP-3 with anaffinity of less than 500 pM.

9D. The antibody or antigen-binding fragment thereof of any ofparagraphs 1-8, wherein said antibody inhibits alternative pathwayactivation in mammalian blood.

10D. A composition comprising the antibody or antigen-binding fragmentof any of paragraphs 1D-9D and a pharmaceutically acceptable excipient.

D. Group II High Affinity MASP-3 Inhibitory Antibodies that Bind One orMore Epitopes Within the SP Domain (1G4)

1E. An isolated antibody, or antigen-binding fragment thereof, thatbinds to MASP-3 comprising:

-   -   (a) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:91 (GYWIE); a HC-CDR2 set forth as SEQ ID NO:93        (EMLPGSGSTHYNEKFKG), and a HC-CDR3 set forth as SEQ ID NO:95        (SIDY); and    -   (b) a light chain variable region comprising a LC-CDR1 set forth        as SEQ ID NO:163 (RSSQSLVQSNGNTYLH), a LC-CDR2 set forth as SEQ        ID NO:165 (KVSNRFS) and a LC-CDR3 set forth as SEQ ID NO:167        (SQSTHVPPT).

2E. The antibody or antigen binding fragment thereof of paragraph 1,wherein the antibody or antigen-binding fragment is selected from thegroup consisting of a human antibody, a humanized antibody, a chimericantibody, a murine antibody, and an antigen-binding fragment of any ofthe foregoing.

3E. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-2, wherein said antibody or antigen binding fragmentthereof is selected from the group consisting of a single chainantibody, an ScFv, a Fab fragment, an Fab′ fragment, an F(ab′)2fragment, a univalent antibody lacking a hinge region and a wholeantibody.

4E. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-3, further comprising an immunoglobulin constant region.

5E. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-4, wherein the antibody or antigen-binding fragment ishumanized.

6E. The isolated antibody or antigen-binding fragment thereof ofparagraph 1, wherein the antibody or antigen-binding fragment thereofcomprise a heavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99%or 100% identical to SEQ ID NO:31 and a light chain comprising at least80%, 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:46 [1G4].

7E. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-6 wherein said antibody binds to human MASP-3 with anaffinity of less than 500 pM.

8E. The antibody or antigen-binding fragment thereof of any ofparagraphs 1-7, wherein said antibody inhibits alternative pathwayactivation in mammalian blood.

9E. A composition comprising the antibody or antigen-binding fragment ofany of paragraphs 1E-8E and a pharmaceutically acceptable excipient.

E. Group III High Affinity MASP-3 Inhibitory Antibodies that Bind One orMore Epitopes within the SP Domain (1E7, 2D7, 15D9, 2F5, 1B11, 2F2,11B6)

1F. An isolated antibody, or antigen-binding fragment thereof, thatbinds to MASP-3 comprising:

-   -   (a) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:109 (RVHFAIRDTNYWMQ), a HC-CDR2 set forth as SEQ ID        NO:110 (AIYPGNGDTSYNQKFKG), a HC-CDR3 set forth as SEQ ID NO:112        (GSHYFDY); and a light chain variable region comprising a        LC-CDR1 set forth as SEQ ID NO:182 (RASQSIGTSIH), a LC-CDR2 set        forth as SEQ ID NO:184 (YASESIS) and a LC-CDR3 set forth as SEQ        ID NO:186 (QQSNSWPYT) [1E7]; or    -   (b) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:125 (DYYMN), a HC-CDR2 set forth as SEQ ID NO:127        (DVNPNNDGTTYNQKFKG), a HC-CDR3 set forth as SEQ ID NO:129        (CPFYYLGKGTHFDY); and a light chain variable region comprising a        LC-CDR1 set forth as SEQ ID NO:196 (RASQDISNFLN), a LC-CDR2 set        forth as SEQ ID NO:198 (YTSRLHS) and a LC-CDR3 set forth as SEQ        ID NO:200 (QQGFTLPWT) [2D7]; or    -   (c) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:132, a HC-CDR2 set forth as SEQ ID NO:133, a        HC-CDR3 set forth as SEQ ID NO:135; and a light chain variable        region comprsing a LC-CDR1 set forth as SEQ ID NO:203, a LC-CDR2        set forth as SEQ ID NO:165 and a LC-CDR3 set forth as SEQ ID        NO:204 [49C11]; or    -   (d) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:137 a HC-CDR2 set forth as SEQ ID NO:138, a HC-CDR3        set forth as SEQ ID NO:140; and a light chain variable region        comprising a LC-CDR1 set forth as SEQ ID NO:206, a LC-CDR2 set        forth as SEQ ID NO:207 and a LC-CDR3 set forth as SEQ ID NO:208        [15D9];or    -   (e) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:98, a HC-CDR2 set forth as SEQ ID NO:99, a HC-CDR3        set forth as SEQ ID NO:101; and a light chain variable region        comprising a LC-CDR1 set forth as SEQ ID NO:169, a LC-CDR2 set        forth as SEQ ID NO:171 and a LC-CDR3 set forth as SEQ ID NO:173.        [2F5]; or    -   (f) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:103, a HC-CDR2 set forth as SEQ ID NO:105, a        HC-CDR3 set forth as SEQ ID NO:107; and a light chain variable        region comprising a LC-CDR1 set forth as SEQ ID NO:176, a        LC-CDR2 set forth as SEQ ID NO:178 and a LC-CDR3 set forth as        SEQ ID NO:193 [1B11]; or    -   (g) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:114, a HC-CDR2 set forth as SEQ ID NO:116, a        HC-CDR3 set forth as SEQ ID NO:118; and a light chain variable        region comprising a LC-CDR1 set forth as SEQ ID NO:188, a        LC-CDR2 set forth as SEQ ID NO:178 and a LC-CDR3 set forth as        SEQ ID NO:190 [2F2]; or    -   (h) a heavy chain variable region comprising a HC-CDR1 set forth        as SEQ ID NO:114, a HC-CDR2 set forth as SEQ ID NO:121, a        HC-CDR3 set forth as SEQ ID NO:123; and a light chain variable        region comprising a LC-CDR1 set forth as SEQ ID NO:191, a        LC-CDR2 set forth as SEQ ID NO:178 and a LC-CDR3 set forth as        SEQ ID NO:193. [11B6]

2F. The antibody or antigen binding fragment thereof of paragraph1(a)-(g), wherein the antibody or antigen-binding fragment is selectedfrom the group consisting of a human antibody, a humanized antibody, achimeric antibody, a murine antibody, and an antigen-binding fragment ofany of the foregoing.

3F. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-2, wherein said antibody or antigen binding fragmentthereof is selected from the group consisting of a single chainantibody, an ScFv, a Fab fragment, an Fab′ fragment, an F(ab′)2fragment, a univalent antibody lacking a hinge region and a wholeantibody.

4F. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-3, further comprising an immunoglobulin constant region.

5F. The antibody or antigen binding fragment thereof of any one ofparagraphs 1-4, wherein the antibody or antigen-binding fragment ishumanized.

6F. The antibody or antigen-binding fragment thereof of paragraph 1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:32 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:47 [1E7].

7F. The antibody or antigen-binding fragment thereof of paragraph 1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:33 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:48 [2D7].

8F. The antibody or antigen-binding fragment thereof of paragraph 1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:34 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:49 [49C11].

9F. The antibody or antigen-binding fragment thereof of paragraph 1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:35 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:50 [15D9]

10F. The antibody or antigen-binding fragment thereof of paragraph 1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:36 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:51 [2F5].

11F. The antibody or antigen-binding fragment thereof of paragraph 1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:37 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:52 [1B11].

12F. The antibody or antigen-binding fragment thereof of paragraph 1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:38 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:53 [2F2].

13F. The antibody or antigen-binding fragment thereof of paragraph 1,wherein the antibody or antigen binding fragment thereof comprises aheavy chain comprising at least 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:39 and a light chain comprising at least 80%,85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:54 [11B6].

14F. The antibody or antigen-binding fragment thereof of any one ofparagraphs 1-13 wherein said antibody binds to human MASP-3 with anaffinity of less than 500 pM.

15F. The antibody or antigen-binding fragment thereof of any ofparagraphs 1-14, wherein said antibody inhibits alternative pathwayactivation in mammalian blood.

16F. A composition comprising the antibody or antigen-binding fragmentof any of paragraphs 1F-15F and a pharmaceutically acceptable excipient.

F. Use of MASP-3 Inhibitory Antibodies for Treatment of AP Diseases

-   -   1. A method of inhibiting alternative pathway complement        activation in a mammal, the method comprising administering to a        mammal subject in need thereof an amount of a composition        comprising a high affinity MASP-3 inhibitory antibody or        antigen-binding fragment thereof sufficient to inhibit        alternative pathway complement pathway activation in the mammal.    -   2. The method of claim 1, wherein the antibody, or antigen        binding fragment thereof binds to MASP-3 with an affinity of        less than 500 pM.    -   3. The method of paragraph 1, wherein as a result of        administering the composition comprising the antibody or        antigen-binding fragment one or more of the following is present        in the mammalian subject:        -   (a) inhibition of Factor D maturation;        -   (b) inhibition of the alternative pathway when administered            to the subject at a molar ratio of from about 1:1 to about            2.5:1 (MASP-3 target to mAb)        -   (c) the classical pathway is not inhibited.        -   (d) inhibition of hemolysis and/or opsonization;        -   (e) a reduction of hemolysis or the reduction of C3 cleavage            and C3b surface deposition;        -   (f) a reduction of Factor B and Bb deposition on an            activating surface;        -   (g) a reduction of resting levels (in circulation, and            without the experimental addition of an activating surface)            of active Factor D relative to pro-Factor D;        -   (h) a reduction of levels of active Factor D relative to            pro-Factor D in response to an activating surface; and/or        -   (i) a reduction of the production of resting and            surface-induced levels of fluid-phase Ba, Bb, C3b, or C3a.    -   4. The method of paragraph 1, wherein the antibody inhibits the        alternative pathway at a molar ratio of from about 1:1 to about        2.5:1 (MASP-3 target to mAb)    -   5. The method of any of paragraphs 1-3 wherein the high affinity        MASP-3 antibody characterized according to any of claims 27A,        25B, 21C, 10D, 9E or 16F.    -   6. The method of any of paragraphs 1-4, wherein the antibody or        antigen binding fragment thereof selectively inhibits the        alternative pathway without affecting the classical pathway        activation.    -   7. The method of any of paragraphs 1-6 wherein the mammal        subject is suffering from, or at risk of developing an        alternative-pathway disease or disorder selected from the group        consisting of paroxysmal nocturnal hemoglobinuria (PNH),        age-related macular degeneration (AMD, including wet and dry        AMD), ischemia-reperfusion injury, arthritis, disseminated        intravascular coagulation, thrombotic microangiopathy (including        hemolytic uremic syndrome (HUS), atypical hemolytic uremic        syndrome (aHUS),thrombotic thrombocytopenic purpura (TTP) or        transplant-associated TMA), asthma, dense deposit disease,        pauci-immune necrotizing crescentic glomerulonephritis,        traumatic brain injury, aspiration pneumonia, endophthalmitis,        neuromyelitis optica , Behcet's disease, multiple sclerosis,        Guillain Barre Syndrome, Alzheimer's disease, Amylotrophic        lateral sclerosis (ALS), lupus nephritis, systemic lupus        erythematosus (SLE), Diabetic retinopathy, Uveitis, Chronic        obstructive pulmonary disease (COPD), C3 glomerulopathy,        transplant rejection, Graft-versus-host disease (GVHD),        hemodialysis, sepsis, Systemic inflammatory response syndrome        (SIRS), Acute Respiratory Distress Syndrome (ARDS), ANCA        vasculitis, Anti-phospholipid syndrome, Atherosclerosis, IgA        Nephropathy and Myasthenia Gravis.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A cloning or expressionvector comprising a nucleic acid encoding complementarity determiningregions (CDRs) of heavy and light chain variable regions of an antibody,or antigen-binding fragment thereof, that binds to human MASP-3, whereinthe heavy chain variable region comprises the amino acid sequence setforth as SEQ ID NO:254 or SEQ ID NO:255 and the light chain variableregion comprises the amino acid sequence set forth as SEQ ID NO:45, SEQID NO:256 or SEQ ID NO:280.
 2. The cloning or expression vector of claim1, wherein the heavy chain variable region comprises SEQ ID NO:254 andthe light chain variable region comprises SEQ ID NO:45.
 3. The cloningor expression vector of claim 1, wherein the heavy chain variable regioncomprises SEQ ID NO:254 and the light chain variable region comprisesSEQ ID NO:256.
 4. The cloning or expression vector of claim 1, whereinthe heavy chain variable region comprises SEQ ID NO:254 and the lightchain variable region comprises SEQ ID NO:280.
 5. The cloning orexpression vector of claim 1, wherein the heavy chain variable regioncomprises SEQ ID NO:255 and the light chain variable region comprisesSEQ ID NO:45.
 6. The cloning or expression vector of claim 1, whereinthe heavy chain variable region comprises SEQ ID NO:255 and the lightchain variable region comprises SEQ ID NO:256.
 7. The cloning orexpression vector of claim 1, wherein the heavy chain variable regioncomprises SEQ ID NO:255 and the light chain variable region comprisesSEQ ID NO:280.
 8. A cell comprising the cloning or expression vectoraccording to claim
 1. 9. A cell comprising: (a) a nucleic acid encodingcomplementarity determining regions (CDRs) of a heavy chain variableregion of an antibody, or antigen-binding fragment thereof, that bindsto human MASP-3, wherein the heavy chain variable region comprising theamino acid sequence set forth in SEQ ID NO:255; and (b) a nucleic acidencoding complementarity regions (CDRs) of a light chain variable regionof an antibody, or antigen-binding fragment thereof, that binds to humanMASP-3, wherein the light chain variable region comprises the amino acidsequence set forth in SEQ ID NO:280.
 10. The cell of claim 9, whereinthe nucleic acid according to (a) and the nucleic acid according to (b)are included in an expression vector in the cell.
 11. The cell of claim9, wherein the nucleic acid according to (a) and the nucleic acidaccording to (b) are included in different expression vectors in thecell.
 12. A method for producing an antibody, or antigen-bindingfragment thereof, that binds to human MASP-3, the method comprisingculturing the cell of claim 8 under conditions and for a time sufficientto allow expression by the cell of the antibody, or antigen-bindingfragment thereof, encoded by the nucleic acid.
 13. The method of claim12, further comprising isolating the antibody, or antigen-bindingfragment thereof.